Study design
Unmatched case–control study was done at three big hospitals in the capital Addis Ababa: Gandhi Memorial Hospital, Tikur Anbessa Hospital and Zewditu Memorial Hospital. The study was completed in 84 participants (42 cases and 42 controls). The required sample size was determined using unmatched case control study sample size calculation formula. Given the lack of concrete information from previous studies on the prevalence of low dietary calcium intake and low serum calcium level among preeclamptic women in similar setting, in order to get representative sample size, we assumed hypothetically identified odds ratio of 4.0 that could yield the representative sample size, a confidence level of 95% and power of 80%. Adjustment was made for population size (less than 10 times from estimated sample size) using finite population correction factor.
Data collection procedures
Cases were recruited based on the current (2019) International Society for the Study of Hypertension in Pregnancy (ISSHP) definition of preeclampsia which is, systolic blood pressure at ≥ 140 mmHg and/or diastolic blood pressure at ≥ 90 mmHg on at least two occasions measured 4 hours apart in previously normotensive women and is accompanied by one or more of the following new onset conditions at or after 20 weeks of gestation: proteinuria (i.e. ≥ 30 mg/mol protein: creatinine ratio; ≥ 300 mg/24 h; or ≥ 2 + dipstick); Evidence of other maternal organ dysfunction, including: acute kidney injury (creatinine ≥ 90 μmol/L; 1 mg/dL); liver involvement (elevated transaminases, e.g. alanine aminotransferase or aspartate aminotransferase > 40 IU/L) with or without right upper quadrant or epigastric abdominal pain; neurological complications (e.g. eclampsia, altered mental status, blindness, stroke, clonus, severe headaches, and persistent visual scotomata); or hematological complications (thrombocytopenia–platelet count < 150 000/μl, disseminated intravascular coagulation, hemolysis); or uteroplacental dysfunction (such as fetal growth restriction, abnormal umbilical artery doppler waveform analysis, or stillbirth) [22]. Cases were selected from the antenatal clinics (ANC), but sometimes followed to the antepartum ward for repeat blood pressure (BP) measurement. Incident cases were chosen to decrease recall bias.
Inclusion criteria for the cases were women aged ≥ 18 years, gestational age ≥ 20 weeks and singleton pregnancy. Those with chronic hypertension, women on anti-hypertensive and anti-convulsant treatment, diabetes mellitus, renal disease, known thyroid and adrenal disease, intrauterine fetal death and those taking antenatal vitamin and mineral supplementation were excluded.
Controls were selected from the hospitals too (source population). Recruiting neighborhood and population controls was not possible in our setting, as the likelihood of finding a healthy non-hypertensive pregnant woman in the neighborhood was unlikely. Any risk factors that could contribute for preeclampsia in the current or previous pregnancies, such as antepartum hemorrhage (APH) and twin pregnancies, were excluded. Controls were women who came for normal ANC follow up, or with obstetric conditions like previous cesarean section scar, or women that could otherwise follow pregnancy at the health centers.
Systematic random sampling technique was used to recruit participants. With the assumption that there could be around 250 cases of preeclampsia in the data collection period, we recruited every sixth patient with preeclampsia and a corresponding number of controls was selected.
Recruitment of cases and controls and coding were done by the principal investigator and the treating physician at ANC and emergency room. Data collectors, who were nurses with Bachelor of Science (BSC) degree, were masked before they took the dietary recall, then unmasked after completing the collection of data related to the dietary recall, as information from patient cards was mandatory to complete the questionnaire. The diagnosis of preeclampsia was made by the consultation physician, and evidences were carefully recorded. Gestational age was determined with the most accurate obstetric estimate, using menstrual history, symphysis–fundal height at the initial antenatal (booking) visit or ultrasound scan, when available. The questionnaire comprised demographic details, booking details of the index pregnancy, general medical and health details, a dietary questionnaire. Blood pressure was measured by trained personnel using a manual pneumatic mercury sphygmomanometer, and utilizing the fifth Korotkoff sound. A repeat measurement was recorded after 4 h to ensure consistency in blood pressure measurement.
Ten milliliters of blood from antecubital fossa was taken, centrifuged, and refrigerated at 2–8 degree centigrade until analyzed. Total serum calcium and ionized serum calcium were determined by using an inductively coupled plasma mass spectrometer. Twenty-four-hour dietary recall was done and calcium content in the meals of the participants were calculated using the food composition table for use in Ethiopia, Part III, developed in 1995–1997 [23].
Data was collected from October 3 to December 25, 2019.
Data analysis
Data was analyzed using SPSS version 25 (IBM, Armonk, NY, USA). Descriptive analyses, Chi-square test, univariate and multivariate logistic regressions were done. Odds ratios (OR), adjusted OR and 95% confidence intervals (CI) were calculated. Pearson/Spearman correlation was performed. P value of less than 0.05 was considered statistically significant. We used t-test statistic to see the association of the variables age, parity, gestational age and body mass index with preeclampsia. The association between dietary calcium intake and total and ionized serum calcium levels, educational level, body mass index, parity, age group and gestational age, income was seen using Chi-square test. Bivariate logistic regression was used to measure the relationship of each covariate with the outcome variable (preeclampsia). Variables that were associated with the outcome variable with p-value of < 0.2 in the univariate analysis and clinically relevant variables that were known to associate with preeclampsia despite their p-values in the univariate analysis were included in to the multiple logistic regression model. Hence, the variables imputed in the multiple logistic regression model were parity, gestational age, total serum calcium level, ionized calcium level and dietary calcium amount. Pearson correlation was used to see if dietary calcium intake, total serum calcium level and ionized calcium level correlated with each other.
Reference (normal) values taken were 2.1–2.55 mmol/l for total serum calcium level, 1.16–1.32 mmol/l for ionized calcium level and Recommended Dietary Allowance (RDA) of ≥ 1000 mg/24 h for dietary calcium intake.
