Anemia during different trimesters of pregnancy and hypertensive disorders are associated with adverse maternal and perinatal outcomes in Bekwai Municipality, Ghana: protocol for a prospective cohort study.


 Background: Data examining whether anemia during different trimesters of pregnancy affects maternal and birth outcomes is lacking. Studies typically look at associations between risk factors and anemia or anemia and birth outcomes but have limited information as to whether birth outcomes differ with different trimesters of anemia. The main objective of this study is to determine whether anemia during different trimesters of pregnancy and hypertension disorders is associated with adverse maternal and perinatal outcomes in Ghana.Methods: This will be a primary analysis of data from a hospital-based prospective cohort study in four selected hospitals in the Bekwai Municipality, Ghana from February 2020 to August 2020. All singleton pregnancy at less than 20 weeks to women with at least one hemoglobin measure during pregnancy will be included. The risk of maternal and perinatal outcomes will be measured. Descriptive statistics will be used to describe the baseline characteristics of our cohort. Baseline characteristics of the two groups will be compared using Pearson Chi-square (χ2) test for categorical variables; the independent t-test and Mann–Whitney U test for parametric and nonparametric continuous variables, respectively. The risk associated with anemia and maternal and perinatal outcomes, using the history of anemia (Hb<11g/dL) in different trimesters will be calculated by logistic regression analysis, conditional on the baseline covariates. Possible confounding variables will be identified from background data, obstetric risk factors, and health behaviors. Multivariate analysis of significant effects (p<0.05) will be based on multiple logistic regression analysis. Confidence intervals will be evaluated at 95%. Data will be coded and examined using the SPSS program IBM version 20.Discussion: The better understanding of whether anemia during different trimesters of pregnancy and hypertension disorders is associated with adverse maternal and perinatal outcomes in Bekwai Municipality of Ghana. This study will establish targets for early intervention and identify risk factors to save and improve the lives of pregnant women and inborn by developing solutions that promote the quality and accountability of health services for the most vulnerable. These findings will be served as a policy document to governments in designing effective programs to curb the increasing prevalence of anemia and its associated health consequences.

problems affecting pregnant women, which differs significantly because of variations in socioeconomic conditions, lifestyles, and health-seeking behaviors across different cultures [30]. Physiologically, plasma volume expands by 25-80% of pre-pregnancy volumes between the second trimester and the middle of the third trimester of pregnancy [31]. This induces a modest decrease in hemoglobin levels during pregnancy. Previous studies show that the best time to investigate any risk factors associated with anemia may be up until 20 weeks of gestation [13].
However, studies focusing on anemia in different trimesters and pregnancy outcomes are scarce, and the findings are inconsistent due to a wide variation in study designs, sample sizes, and populations. Furthermore, not all studies reported adjustments for confounders which could affect the final outcome, such as the timing of hemoglobin assessment[32] and possibly over or underestimate risks. For instance, Rohilla and colleagues [33], conducted a study using a hemoglobin concentration of < 7 g/dl to determine the maternal and perinatal outcome in patients with severe anemia in pregnancy. They reported higher proportions of hypertensive disorders, adverse maternal and perinatal outcomes. Hemoglobin levels measurement is a standard during prenatal visit and it is used to evaluate the physical status and anemia [34]. According to the World Health Organization [35], anemia is diagnosed when a blood test shows a hemoglobin value of less than 11.0 g/dL in pregnant women. However, few studies to date have assessed the effects of anemia during different trimesters and adverse maternal-perinatal outcomes.
Therefore this study will seek to evaluate whether associations exist between anemia during different trimester, hypertensive disorders, and adverse maternalperinatal outcomes in the study area.

Study objective
The main objective of this study is to determine whether anemia during different trimesters of pregnancy and hypertension disorders is associated with adverse maternal and perinatal outcomes in Bekwai Municipality, Ghana

Specific objectives
To determine whether anemia during different trimesters of pregnancy is associated with adverse maternal and perinatal outcomes in the study population.
To determine whether hypertension disorders in pregnancy is associated with adverse maternal and perinatal outcomes in the study population.

Study Design
This study will be conducted using a hospital-based prospective cohort study aimed at enrolling pregnant women in the first trimester to identify anemia or no anemia at the baseline. The selected subjects will be followed up to delivery to determine whether anemia during different trimesters of pregnancy and hypertension disorders is associated with the risk of adverse maternal and perinatal outcomes in Bekwai Municipality, Ghana.

Study settings
This study will be conducted in four selected hospitals across the Bekwai Municipality for the last 12 months prior to the census night was 2,897. The data further indicates that the total fertility rate was approximate 3.5.

Sample size considerations
The sample size will be calculated using a PASS for determining proportions for the study population. Where N 1 =the first sample size, Power (1-Beta )=0.90, Inspection performance is 90% Alpha (Significance Level)=0.05, Inspection level is 0.05N2 (Sample Size Group 2): Use R, Two groups of sample ratio coefficient R (Sample Allocation Ratio): 1, Two groups of cases Equivalent R 1 (Ratio|H 1 =P 1 /P 2 )= 0.5, RR value is 2P 2 (Control Group Proportion)=0.23, Control event rate is 23%, Alternative Hypothesis (H 1 )= Two-Sided, Alternative Hypothesis (Two Sides Test)=Test Type= Z Test ( Pooled). Then, the total sample size for exposed/unexposed will be 225 each for the group. Hence the total sample size for exposed/unexposed with a 10% continuity correction will be approximately 250 each for group and total of 500.
Therefore, for a normally distributed continuous outcome, it is possible to detect with a type I error of 5%, two-sided significance level (1-alpha) of 95% confidence interval, and a type II error of 10% with 90% power (1-beta, % chance of detecting).

Inclusion criteria
Pregnant women who had no bleeding at the time of recruitment will be included. All pregnant women aged 15-49 years who has given consent personally with anemia or without will be included, regardless of whether or not they had taken iron food consumption will be undertaken from enrolled pregnant women. This food frequency questionnaire will help to assess the die consumed by women in district Municipality. Besides, information of occupation and income status of the husband and be examined.

Hypertensive disorders in pregnancy
Following the course of the pregnancy, the following information will be collected from exposed and non-exposed. Blood pressure will be measured in each trimester with the validated Omron RCHS automated digital sphygmomanometer. All participants will be seated in an upright position with back support for 5 min. An appropriate cuff will be placed around the non-dominant upper arm, which will be supported at the level of the heart, with the bladder midline over the brachial artery pulsation. The mean value of two blood pressure readings will be documented over a 2min interval. Information on chronic hypertension superimposed preeclampsia, preeclampsia or eclampsia, and gestational hypertension will be derived from maternal antenatal registries.

Post-delivery data collection
All participants will be followed-up and will be also contacted twice during their pregnancy over the telephone to enquire about their well-being. Post-delivery data will be collected involving in daily identification of all the women who had delivered during the previous 24 h in the hospital by the support of station nurse. Baseline data will be extracted from patients' folders, admission and discharge registers at the labor wards. After the initial daily baseline data extraction on all the parturient incomes (fetal distress, neonatal intensive care (NICU) admission, stillbirth, early neonatal death, low birth weight, small for gestation age, birth weight, macrosomia, preterm delivery, APGAR score < 7 at 1 min and APGAR score <7 at 5 min) and mothers outcomes (transfusion, hypertensive disorders, postpartum hemorrhage, gestational diabetes, and maternal mortality) will be identified and their folder numbers recorded. Post-delivery data collection will be completed by August 30, 2020.

Data management and quality control
The data collectors and key informants will be staff midwives nurses and one research assistant supervised by the principal investigator. Training and practical demonstrations on the interview techniques and measurement procedures will be given to data collectors for two consecutive days. A pilot study will be done on 20 ANC attendants over a period of 5days to determine the appropriateness and completeness of the questionnaire for the study. Information that will be retrieved from the ANC books will be confirmed by the principal investigator for its consistency. After informed consent has been obtained, eligible participants will be interviewed using a semi-structured questionnaire. Maternal records will be reviewed on specific items related to the study. Data collected will be then entered into a safe computerized database using a unique code to ensure data accuracy.
The databases will have patient-identifiable information attached such as name and address, and each patient will have an anonymized study ID. A copy of the anonymized database (without a name, address) will be sent to SPSS for data analysis. The patient identity will be protected and only aggregate summary data will be released publically (e.g., in the form of a peer-reviewed publication),

Maternal health outcomes
Data will be available to performed analyses for the following selected maternal health outcomes: (1) transfusion, (2) hypertensive disorders, (3) postpartum hemorrhage, (4) and gestational diabetes and (5) maternal mortality. The results for these outcomes will be presented by (1) time period: first, second, and third trimesters anemia; (2) cutoff: ≤7.0 g/dL, 7.0-9.9 g/dL, 10-10.9 g/dL, and ≥11.0 g/dL (3) overall estimate of anemia throughout pregnancy will be analyzed. Results of the association between prenatal anemia and maternal outcomes will be summarized in tables.

Statistical analysis
The distribution of the study variables such as sociodemographic factors, nutritional factors, genetic factors, behavioral factors and obstetric factors of exposed and non-exposed will be calculated using means with standard deviations for normal continuous variables and frequencies and percent for categorical variables.
Univariate comparisons will be performed using the Pearson Chi-square (χ 2 ) for categorical variables; the independent t-test and Mann-Whitney U test for parametric and nonparametric continuous variables, respectively when the quality of variances is satisfied. Binary logistic regression analysis will be used to evaluate the risk associated between anemia and adverse maternal and perinatal outcomes, using history of anemia (Hb<11g/dL) in different trimester by calculating the odds of different exposures and non-exposed, taking into account, when the interaction terms are observed followed by multiple logistic regression and adjusting for potential confounders. Similar models will be used to examine the associations between hypertensive disorders and adverse maternal and perinatal outcomes. The variables for which p-value turns out to be less than 0.2 will be included in the multivariable analysis. In multivariate analysis of significant effects (p<0.05) will be based on multiple logistic regression analysis. Confidence intervals will be evaluated at 95%. During this analysis interaction between the variables will be assessed. This analysis will also be helpful in identifying the potential confounders such as sociodemographic factors, nutritional factors, genetic factors, behavioral factors and obstetric factors related to the study. In addition, we will also explore the role of first-trimester anemia only, second-trimester anemia only, thirdtrimester anemia only, first-trimester anemia and second-trimester anemia only, first-trimester anemia and third-trimester anemia on, second-trimester anemia and third-trimester anemia and adverse maternal and perinatal outcomes. Data will be coded and examined using the SPSS program IBM version 20.

Discussion
The better understanding of whether anemia during different trimesters of pregnancy and hypertension disorders is associated with adverse maternal and perinatal outcomes in the Bekwai Municipality of Ghana. This will, in turn, decrease the chances of adverse maternal and perinatal outcomes associated with anemia. This study will establish targets for early intervention and identify risk factors to save and improve the lives of pregnant women and inborn by developing solutions that promote the quality and accountability of health services for the most vulnerable population of Ghana. These findings will be served as a policy document to governments in designing effective programs to curb the increasing prevalence of anemia and its associated health consequences.

Strengths and weaknesses of the study
The primary strength of this study will be the in-person collection of exposure and outcome measures using research standard procedures that will be conducted by trained and expert staff. Another is the wealth of covariate information that will be collected, which will helps to minimize confounding. Weaknesses may include the loss to follow-up over time to miscarriages, maternal transfers from the study area and other others reasons not specified.

Ethical approval and Consent to participant
There will be no major ethical issues in this study as it will be carried out within the confines of routine antenatal care. Those found to be anemic will follow the routine protocol for anemia treatment. Approval is given by institutional review board (IRB), human subjects committee of Central South University, China. Administrative permission will be granted from the Bekwai Municipal District Health Management Team. Informed consent will be obtained from the mothers before participating in this study. Informed consent will be obtained from all new antenatal women who will consent to the study.. The objective of the study will be explained and each study