Examining the Effect of Education on Dietary Calcium Intake in Reducing Blood Pressure Variability Among Pregnant Mothers in Tigray Region, Northern Ethiopia, Two Arm, Randomized Control Trail Parallel Design

Background: Investigating effects of calcium diets on blood pressure can contribute to development of diet based recommendations for health. Epidemiologic data suggest contradicting evidence relationship between dietary calcium intake and pregnancy-induced hypertension. So, this study aimed to determine the effect of calcium nutrition education on blood pressure variability among pregnant mother. Method: Single center, two-arm, main individual randomized trial parallel design; single blinded was conducted among 415 pregnant mothers. Bi-variable tabulations were computed to identify the distributions of the outcome variables by selected background characteristics. We estimated a multilevel model that assessed the relation of individual and community level factors (xed effects) as well as community level random effects. All analysis was conducted using STATA 14 software. Result: Blood pressure variability during pregnancy after 28 gestational age was signicantly associated with weekly calcium nutrition education [AOR=0.38; 95% CI: 0.19, 0.5], pregnant mothers who attained secondary school [AOR=0.21, 95%CI: 0.20, 0.70], employed pregnant women [AOR=9.05; 95% CI: 1.95, 14.02] , Antenatal Care [AOR=1.82; 95% CI: 1.01, 2.22], supplemented iron/folic acid [AOR=6.32; 95% CI: 1.09, 36.59], food craving [AOR=0.78; 95% CI: 0.20, 0.98], reading newspaper [AOR=9.05; 95% CI: 1.95, 14.02], place of residence [AOR=2.11; 95% CI: 1.36, 3.26]. Conclusion: Individual level factors (Calcium nutrition education, maternal educational status, maternal occupation, Antenatal care during last pregnancy and current pregnancy, iron/folic acid supplementation, food craving, dietary calcium level and reading newspaper) and community level factors (place of residence) were signicant predictors of blood pressure variability. During antenatal visits, pregnant women should be made aware of some dietary practices which are harmful during pregnancy, and increase education regarding the benet of adequate nutrition. Trail Registration: PACTR,

about one out of every 10 maternal deaths in Africa and Asia have been attributed to pre eclampsia and eclampsia [6].
WHO recommended Calcium supplementation during early pregnancy for population with poor dietary calcium intake to prevent pregnancy related hypertension [2,4]. In Ethiopia dietary calcium intake is too law, which accounts 88.4% women have inadequate dietary calcium intake and also in Tigray the average mean intake of women of child bearing age was estimated 296.67 ± 1.04 mg/day, which is below the recommended EAR for women [7].
However, in poor counters availability and affordability of calcium are the main challenge to implement the recommendation [2,4]. Due to this other studies recommend that healthy dietary intake can meet micronutrient needs of pregnant women in developing countries [8,9]. Thus, this trail aimed to determine the effect of education on dietary calcium intake in decreasing variability of blood pressure in pregnant mothers.

Methodology Study Area, period and Trail design
Single center, two-arm, main individual randomized trial parallel design, single blinded including 12-16 weeks follow up was conducted in selected district of Tigray Region from June to September 2019.
Tigray is one of the 9 Regional states of Ethiopia and Mekelle is the capital city of the Region which is located 783 km North of Addis Ababa which is capital city of Ethiopia. Tigray Region, which has about one million hectares of arable land in total, is also successfully expanding its agriculture extension program. In Tigray, bread is one of the main foods. Two of the more common varieties are thin, pancakelike bread preferred by most people and a dense, disk-shaped loaf of baked whole wheat bread and is made from many kinds of cereal grains (wheat, barley, etc.). A variety of tsebhi (spicy stews) are eaten with the bread.

Population
All greater than 28 weeks gestational age pregnant mothers in Tigray region were the source population and all randomly selected pregnant mothers from the selected districts were study population.

Eligibility criteria
Pregnant women who had a mean systolic BP from 120 to 140 mmHg and a diastolic BP from 80 to 90 mmHg, based on six readings at two screening visits were included to the study and Mothers with a systolic BP ≥140 mmHg or a diastolic BP ≥90 mmHg or that were taking antihypertensive medications was excluded. In addition, persons with a self-reported history of clinical cardiovascular disease (CVD), cancer, chronic kidney disease, body mass index (BMI) ≥30 kg/m2 were excluded from the study.

Sample size determination
The sample size for the study was calculated from a study which indicates maternal death due to hypertensive disorder was 20%. Sample size was computed to detect minimum of 10% additional reduction in intervention as compared to the control arm with the following assumptions: level of signi cance 5%; Power 80%, lost to follow up 10%. The required sample size was 415 subjects in all groups. The total duration of the intervention was 12-16 weeks.

Trail intervention
Dietary intake was assessed at baseline and in every weekly visit to household using an interactive 24hour recall method. After completing the baseline assessments (described further in study assessments) mothers were randomly assigned to either the education on dietary intake of calcium containing foods or the control group. The randomization was 1:1 for the 2 groups and was performed using simple randomization computer generated codes for each individual. For pregnant mothers in the calcium nutritional education intake group, a target calcium intake was calculated on the basis of 1200 milligram per day baseline, and the importance of meeting this target intake was explained to mothers. An estimate of current dietary intake was made from an interactive 24-h food recall; education was provided about the intake in relation to the target intake, and mothers were given a dietary plan to meet the target intake. Pregnant mothers were instructed to consume these foods per serving required to meet the target.
Pregnant mothers were contacted house to house 3 times per week to assess progress and to provide the necessary feedback. Blood pressure was measured every month until the end of the intervention period. For pregnant mothers randomly assigned to the control group, general advice was given to address any major dietary imbalance identi ed from the interactive 24-h food recall at baseline. They were instructed to increase their food intake as they felt able but were not given a speci c plan for dietary intake.

Measurement of Blood Pressure
Blood pressure was measured once per week per subject using an aneroid sphygmomanometer (Marshall Electronics, Inc., Clayton Division, Skokie, Ill.). Readings was recorded three times with 1 min between each reading. Participants was allowed a 10-min rest period before readings taken. All participants maintained a sitting position while blood pressure was measured in the non-dominant arm. A measurement was made at the same time of day by the same investigator, who was trained and experienced in the recording of blood pressure.

Randomization and Blinding
Each participating centre was assigned a special code generated by the computer using the simple randomization. The randomization was kept by the PI. The assessor for clinical outcomes was blinded to the randomization status. The research assistants who perform the intervention did not know the assessment result. After the eligibility assessment, blood pressure and dietary recall was conducted at the baseline (after completion of informed consent), at 4th, 8th and 12th weeks and every visit respectively.

Study outcome
The primary outcome of interest was the BP variability between the study groups and Incidence of pregnancy induced hypertension were secondary outcome.
Explanatory variables: such as age, marital status, religion, educational level, occupation, monthly house hold income, family size, ANC visit, housing characteristics, household food insecurity, diversity of diet.

Data collection procedures and quality Assurance
The survey was community based structured and semi-structured, pre-tested, interviewer administered questionnaire mainly with closed ended questions was interviewed to pregnant mothers. The questionnaire was used to collect data through face-to-face interview on socio-demographic and dietary calcium intake. The questionnaires have adapted from different studies considering the local situation of the study area and contextualized to the study based on the relevant variables to be used. Data collectors provided a four days training on the data collection process. The research tool was rst developed in English and then translated to the local language Tigrigna. The nal version of the questionnaire was both in Tigrigna and English. Tigrigna version of the questionnaire was used for data collection to ensure clear understanding of the interviewee and respondents. To maintain quality and to estimate the time required collecting data, a pre-test was done on 21 individuals in nearby community to the intervention area to check the feasibility of the data collection process. Questionnaires were revised, as necessary, based on the pre-test and time required to ll one questionnaire was determined. During the study data collection, questionnaires were checked for its completeness and consistency by supervisors and principal investigators. Data that have entered in Epi data version 3.7 was checked for its accuracy by the principal investigators.

Data management and analysis
Blood pressure variability was compared between the intervention groups. Bi variable tabulations were computed to identify the distributions of the outcome variables by selected background characteristics.
We estimated a multilevel model that assessed the relation of individual and community level factors ( xed effects) as well as community level random effects. Facility and community variables were considered as 'community level' variables in the study. Multilevel analysis was used to account for the hierarchical nature of the data. A two-level multilevel logistic regression model was estimated. The model consisted of two sub models at level 1 and level 2 (i.e., individuals (level 1) were nested within communities (level 2). A two-level multilevel model for a dichotomous outcome uses a binomial sampling and a logit link. We estimated four models. The rst model was an empty model containing no covariates, but decomposed the total variance into individual and community components. The second model included individual characteristics. The third model contained only the community characteristics and this allowed the assessment of the relation of the community variables to the outcome variable. The nal model contained explanatory variables at both the individual and community levels and allowed the assessment of the net effect of community variables over and above the individual variables. The variables were retained in each of the models if the variance component was signi cant (p<0.05) or if they were important demographic variables. In all the estimated models, xed effects were expressed as odds ratios (OR), while the random effects were expressed as variance partition coe cient (VPC) and proportional change in variance (PCV). Data was analysis using STATA 14.

Ethical consideration
Ethical clearance was obtained from Tigray health research institute, and an o cial support and permission letter was obtained from Tigray Regional Health Bureau. Written informed consent was assuring from the study participants, after explaining the purpose and signi cance of the study and they was assured that they had the right to withdraw from the study at any stage. Interview was conducted after receiving their consent. Con dentiality of the data/information was secured by assigning unique code to each of the participants and will not be used for other purpose.

Result
Demographic and socio-economic characteristics A total of 415 early third trimester pregnant mothers were recruited. Of these, 203 were in the intervention group and another 212 in the control group with 100 % response rate. The mean ages (±SD) of the mothers were 39(±15) years. Out of the total mothers interviewed, three fourth 305 (73.5%) belongs to the age group of 20-34 years. Most of the mothers 393(94.7%) were currently in marital union and 395(95.2) them were orthodox religion believer. Six out of ten 254 (61.2%) of the mothers were rural residence, greater than three-fourth of them had less 5 family members 320(77.1%). More than one third 320 (41.9%) of the mothers and almost half of the husbands 196 (47.2%) were illiterate. Majority of the maternal occupational 333(80.2%) were housewife and more than half paternal occupation 234 (56.4%) were farmers. One fth of the mothers 86(20.72%) had middle wealth quintile [ Table 1]. Environmental characteristics Around four out of ten 182(43.9%) households did not possess toilet and out of the owners of toilet 112(48.1%) were pit latrine without slab [ Table 2].   Majority of the mothers did not read a newspaper or magazine 275(66.3%) and More than half 249(59.8%) of the mothers did not listen radio and more than one third 284(68.4%) did not watch television [ Table 5]. Factors affecting Blood pressure variability among groups Table 6 shows the results of mixed effect logistic regression analyses examining the effect of calcium nutrition education among pregnant women's individual characteristics and community-level factors in pregnant women in blood pressure uctuation. Model 1, the empty model, includes only random intercept to capture between-cluster variability. In this model, 16% of the total variance in the odds of in blood pressure uctuation was accounted for by between-cluster variation (ICC=0.12, The ICC was computed in each successive model to understand the relative effects of individual-level and community level factors on women in blood pressure uctuation. Similarly, the ICC was computed in each successive model to understand the relative effects of individual-level and community level factors on calcium nutrition education to in blood pressure uctuation. The between-cluster variability over successive models, from 12% in the empty model, to 9.7% in the individual-level only model, 8.9% in the community-level only model, and 8.3% in the combined model [ Table 6]. Showing that variation in the blood pressure variability was explained best by the inclusion of both individual level characteristics.
The proportional of change in model in variance indicated the addition of predicators to the empty model better explained pregnant women calcium nutrition education change in blood pressure uctuation.

Discussion
The primary objective of this research was to examine the effect of calcium nutrition education on blood pressure variability among pregnant mothers, and accordingly pregnant mother who got nutrition calcium education had decreased blood pressure variability. And other signi cant factors that affect blood pressure variability among pregnant mothers were individual level factors (maternal educational status, maternal occupation, ANC during their last and current pregnancy, iron/folic acid supplementation, food craving, calcium intake and reading news paper) and community factors (place of residence).
Calcium nutrition education showed signi cant association with blood pressure variability among pregnant mothers. This result indicates that mothers who had knowledge regarding what to feed that have effect on blood pressure variability had less likely of blood pressure variability. A study done in Ethiopia in support of this showed that pregnant mothers who has no awareness on risk hypertension were more likely develop PIH as compared to their counter parts [10]. This result indicates that if pregnant mother are educated regarding calcium nutrition from previous evidence which has been proposed that low-calcium intake may increase blood pressure by stimulating either parathyroid hormone or renin release, increasing intracellular calcium in vascular smooth muscle and leading to vasoconstriction. Calcium supplementation may reduce parathyroid release and could reduce smooth muscle contractility.
It could also reduce uterine smooth muscle contractility or increase serum magnesium levels and thus prevent preterm labour and delivery [11]. And also other meta-analysis result conducted in United Kingdom showed that among interventions, those based mainly on diet showed a signi cant reduction in pre-eclampsia by 33%, compared with the controls [12].
Other nding were maternal education has signi cant association with blood pressure variability speci cally educated pregnant mothers had lower blood pressure variability. This result is similar with study done in Netherland that showed that women with relatively low levels of education had a higher risk of gestational hypertension than women with a high level [13]. This implies that if we educate mothers on nutrition there might decrease pregnancy induced hypertension.
The study revealed that maternal occupation was signi cantly affecting blood pressure variability among pregnant mothers. Previous studies have found mixed outcomes. This study was in line with study done in Bangladesh [14]. However this nding was inconsistent with study done in Taiwan [15]. Employed pregnant mothers are at risk of blood pressure variability this could be due to one of the suggested mechanism through which physically demanding work and psychological stress could lead to hypertensive disorders during pregnancy is an increased utero placental vascular resistance which follows physical exertion. Physically demanding work may cause an increase in catecholamine levels, which may lead to a decreased uterine blood ow and therefore may induce PIH and preeclampsia. It has also been suggested that part of the excess catecholamine release is due to an overactive sympathetic nervous system [16]. Whatever this could support the nding; however, further research is warranted to con rm these negative ndings.
Another signi cant predictor for blood pressure variability was Antenatal care follow-up during the last pregnancy and current pregnancy. Antenatal care (ANC) is a care of women during pregnancy by skilled health care providers. The components of ANC include: early high risk screening, prevention and care of pregnancy-related complications, including PIH, and provision of health education and health promotion. With adequate ANC (≥ 4 times), pregnant women would be monitored and have better pregnancy outcomes and a reduction in complications. This study is supported by study done in Thailand [17], USA [18]. Pregnancy induced hypertension is not a totally preventable disease but maternal and foetal Complication due to PIH can be halted at mild stage by quality Antenatal care with good outcome through controlling blood pressure variability.
The other main exposure variable used in our study was self-reported consumption of iron and folic acid supplementation during pregnancy. Iron/folic supplementation had signi cant association with blood pressure variability among pregnant mothers. This nding is consistent with the study done in India which indicates the likelihood of reporting Preeclampsia or eclampsia symptoms was lower among those mothers who consumed iron and folic acid supplementation for at least 90 days during their last pregnancy [19]. Iron plays a signi cant role inter alia in: Oxygen transport, in the production of ATP, in the synthesis of DNA, in preserving the function of mitochondria and in protecting cell structures against oxidative damage, in the activity of numerous enzymes, as well as in cell growth and proliferation. Iron plays a catalyzing role in the production of reactive oxygen species in the Fenton and Haber-Weiss reactions. The role of iron in the development of the placenta is not fully understood, but the discovery of the hypoxia-inducible factor (HIF) and its regulatory mechanisms also drew attention to the importance of iron. Another systematic review and Meta analysis done in china showed that iron de ciency correlates with some complications of pregnancy [20].
Food craving was another predictor for blood pressure variability at individual level. This was consistent with study done in Ghana which revealed that speci cally geophagia was associated with pregnancy diastolic blood pressure variability [21]. Cravings in women have been shown to increase in frequency and intensity at two distinct times: during the perimenstrum (i.e., a period of about eight days around the onset of menstruation) and in pregnancy [22].
Dietary calcium intake was one of the signi cant factors associated with blood pressure variability. Low calcium intake has been hypothesized to cause increase in blood pressure by stimulating the release of parathyroid hormone and/or renin which leads to increased intracellular calcium concentration in vascular smooth muscle cells and causes vasoconstriction. Whatever different research nding [23,24] showed that calcium supplementation had signi cant effect on decreasing blood pressure variability and also this study indicates that if we educate mother on calcium nutrition there might be increase consumption of calcium so might lead to decreased blood pressure variability.
The other signi cant factor was reading newspaper, this might be due to if pregnant mother are reading newspaper they could get information concerning their pregnancy nutrition then after they may have knowledge what feed and regarding the complication during pregnancy. Health literacy is the degree to which individuals have the capacity to obtain, process, and understand basic health information and services needed to make appropriate health decisions. It comprises a complex set of reading, listening, analytical, decision-making skills, and the ability to apply these skills to health problems. Low health literacy during the perinatal period, and speci cally about preeclampsia, may contribute not only to women's poor understanding of pregnancy and potential fetal health issues, but also to delayed care and poor health outcomes.
Regarding the community level factors place of residence was one of the signi cant factors affecting blood pressure variability. Urban residents were at risk of blood pressure variability. However a study done in Ethiopia, tigray showed that rural residents were at greater odds of suffering from hypertensive disorders [25]. The difference could be due to the study design used because they are different.

Conclusion
Individual level factors were signi cantly associated with blood pressure variability. Individual level factors (Calcium nutrition education, maternal educational status, maternal occupation, Antenatal care during last pregnancy and current pregnancy, iron/folic acid supplementation, food craving, dietary calcium level and reading newspaper) and community level factors (place of residence) were signi cant predictors of blood pressure variability among pregnant mother after 28 gestational age. During antenatal visits, pregnant women should be made aware of some dietary practices which are harmful during pregnancy, and increase education regarding the bene t of adequate nutrition. Nutrient supplementation should be administered to pregnant women, especially those living in low socioeconomic areas to supplement dietary intakes. A prospective cohort study should have to be conducted using blood biomarkers of nutrients to properly determine the role of nutrients in the development of hypertension in pregnancy. Calcium nutrition education should have to be given through the health extension workers after developing guideline and Regional food composition Availability data and material The datasets used and/or analyzed during the current study is available from the corresponding author on request.

Competing interest
The authors' declare that they have no con ict of interest.

Funding
This research is not funded by any organization