Non-inferiority of low-dose compared to standard high-dose calcium supplementation in pregnancy: study protocol for two randomized, parallel group, non-inferiority trials in India and Tanzania

Background: Hypertensive disorders of pregnancy are important causes of maternal morbidity and mortality, as well as preterm birth, the leading cause of death for children under 5 years globally. The World Health Organization currently recommends that pregnant women receive high-dose calcium supplementation (1500–2000 mg elemental calcium) for prevention of preeclampsia in populations with low dietary calcium intake. Trials of low-dose calcium supplementation (< 1000 mg elemental calcium/day) during pregnancy have also shown similar reductions in the risk of preeclampsia; however, no trials to date have directly compared low-dose to the standard high-dose calcium supplementation. Our objective is to assess the non-inferiority of low-dose as compared to standard high-dose calcium supplementation in pregnancy. Methods/design: We will conduct two independent trials in Bangalore, India (n = 11,000 pregnancies), and Dar es Salaam, Tanzania (n = 11,000 pregnancies). The trial designs are individually randomized, parallel group, quadruple-blind, non-inferiority trials of low-dose calcium supplementation (500 mg elemental calcium/day) as compared to standard high-dose calcium supplementation (1500 mg elemental calcium/day) among nulliparous pregnant women. Pregnant women will be enrolled in the trial before 20 weeks of gestation and will receive the randomized calcium regimen from randomization until the time of delivery. The co-primary outcomes are (i) preeclampsia and (ii) preterm birth; we will test non-inferiority of the primary outcomes for low-dose as compared to the standard high-dose supplementation regimen in each trial. The trials’ secondary outcomes include gestational hypertension, severe features of preeclampsia, pregnancy-related death, third trimester severe anemia, fetal death, stillbirth, low birthweight, small-for-gestational age birth, and infant death. Discussion: The trials will provide causal evidence on the non-inferiority of low-dose as compared to the standard high-dose supplementation in India and Tanzania. A single tablet, low-dose calcium supplementation regimen may improve individual-level adherence, reduce programmatic costs, and ultimately expand implementation of routine calcium supplementation in pregnancy in populations with low dietary calcium intake. Trial registration: ClinicalTrials.gov identifier: NCT03350516; registered on 22 November 2018. Clinical Trials Registry—India identifier: CTRI/2018/02/012119; registered on 23 February 2018. Tanzania Medicines and Medical Devices Authority Trials Registry identifier: TFDA0018/CTR/0010/5; registered on 20 December 2018. We will conduct two independent trials in India and Tanzania. Each will be an individually randomized, parallel group, non-inferiority trial of low-dose 500 mg calcium supplementation versus standard high-dose 1500 mg calcium supplementation for nulliparous pregnant women. The eligibility criteria, intervention, follow-up methods, and outcome definitions have been unified between the two trials; however, each trial will be conducted, analyzed, and reported separately. The first participant was enrolled in the India trial on November 30, 2018, and follow-up data collection is planned to continue through August 2022. The first participant was enrolled in the Tanzania trial on March 22, 2019, and follow-up data collection is planned to continue through November 2022. This trial protocol was written following the Standard Protocol Items: Recommendations for Interventional trials (SPIRIT) checklist (see Additional file 1). The trial in India (clinicaltrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT03350516","term_id":"NCT03350516"}}NCT03350516; Clinical Trials Registry—India identifier: CTRI/2018/02/012119) will be conducted in urban Bengaluru at 11 health facilities that provide antenatal care within the Greater Bengaluru Metropolitan Corporation system (locally referred to as the Bruhat Bengaluru Mahanagar Palike (BBMP)) (clinicaltrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT03350516","term_id":"NCT03350516"}}NCT03350516 and Clinical trials registry—India identifier: CTRI/2018/02/012119). The trial in Tanzania will be conducted in urban Dar es Salaam at 6 health facilities that provide antenatal care (clinicaltrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT03350516","term_id":"NCT03350516"}}NCT03350516 and Tanzania Medicines and Medical Devices Authority Trials Registry identifier: TFDA0018/CTR/0010/5). The trial flow diagram is presented in Fig. ​Fig.1.1. The India and Tanzania trials have the same eligibility criteria for participant enrollment. The inclusion criteria are (a) nulliparous pregnancy, (b) attend first antenatal care visit at the study clinic, (c) less than 20 weeks gestation based on last menstrual period at the time of randomization, (d) ≥ 18 years old, (e) intend to stay in study area until 6 weeks postpartum, and (f) provide written informed consent. Pregnant women will be excluded if they have a (a) history, signs, or symptoms of nephrolithiasis, (b) prior diagnosis of parathyroid disorder or thyroidectomy, or (c) disease that requires digoxin, phenytoin, or tetracycline therapy. Trained research staff will assess eligibility criteria for all participants presenting to the antenatal care clinic and ask potential participants for written informed consent for trial enrollment. Participants will also be asked for their written consent for use of their data and biological specimens (if applicable) in ancillary studies. During the conduct of the trial, the investigators will monitor monthly enrollment targets for the trials and may consider including additional study clinics to increase enrollment if recruitment is slower than expected. Trial flow diagram for the India and Tanzania trials The India and Tanzania trials will assess the non-inferiority of a low-dose calcium supplementation regimen (500 mg elemental calcium/day) as compared to the standard high-dose calcium supplementation regimen (1500 mg elemental calcium/day). The trial regimens will be taken by pregnant women from randomization to the time of delivery (no postnatal supplementation). The standard high-dose 1500 mg calcium supplementation regimen was selected to align with WHO recommendations [10]. The low-dose calcium supplementation regimen was selected to align with low-dose calcium supplementation regimens used in prior low-dose calcium trials [9]. Both randomized groups will take three regimen tablets per day to have identical pill burden in both randomized groups. The trial regimen is manufactured by Influx Healthcare (Maharashtra, India). Women in both randomized groups will receive a regimen box containing a 35-day supply of trial regimen in blister packs. Each blister pack will contain a 7-day supply of trial regimen (7 × 3 tablets per blister pack) with columns indicating morning, midday, and evening tablets. A diagram of the regimen blister pack for each trial is presented in Additional file 2. Participants in both groups will be counseled to take one trial regimen tablet in the morning, one in the midday, and one in the evening with meals and to take iron-containing prenatal supplements provided as standard of care several hours apart from the calcium trial regimen. Standard high-dose calcium group (1500 mg elemental calcium/day): Pregnant women will be provided and counseled to take three regimen tablets daily with each containing 500 mg elemental calcium as calcium carbonate supplements daily (total of 1500 mg elemental calcium). In India, daily vitamin D3 supplementation (250 IU) is recommended to be taken with calcium supplements and therefore each tablet in the standard high-dose calcium group will also contain 83.3 IU of vitamin D3 for a total of 250 IU vitamin D3 daily [14]. In Tanzania, the supplements in the standard high-dose calcium group will not contain vitamin D3. Low-dose calcium group (500 mg elemental calcium/day): pregnant women will be provided and counseled to take three regimen tablets each day with the first morning pill containing 500 mg elemental calcium as calcium carbonate, while the second (midday) and third (evening) tablets will be placebo supplements (total of 500 mg elemental calcium per day). The placebo tablets will be identical in all ways as compared to the calcium supplements (appearance, color, shape, size, weight, taste). In India, each of the three daily regimen tablets in the low-dose calcium group will also contain 83.3 IU of vitamin D3 for a total of 250 IU vitamin D3/day. Therefore, the daily vitamin D3 dose will be identical between standard high-dose and low-dose calcium groups in India. In Tanzania, the supplements in the low-dose calcium group will also not contain vitamin D3. Multiple strategies will be used to improve adherence to interventions in India and Tanzania. At both sites, research staff will take a pill count at each study visit by counting the number of unconsumed tablets returned in the regimen blister packs. Study staff will counsel participants on taking the trial regimen and provide advice on overcoming barriers. At the India site, community health workers (accredited social health activists (ASHAs)) will also be engaged to promote regimen adherence during their routine home visits. At the Tanzania site, biweekly text message reminders for adherence and upcoming study visits will be sent. At both the India and Tanzania trial sites, reminder telephone calls will also be made prior participants’ scheduled visits by the research team and if the subject anticipates that she will not be able to attend the clinic visit or does not attend the clinic visit as planned, a home visit will be made by research staff. The research staff will provide the regimen, take pill counts, and conduct clinical assessments (including blood pressure and proteinuria) at home visits. Participants may withdraw from the study at any time. In addition, study investigators or study physicians can decide that a participant should not continue to take the trial regimen or participate in the study if there are safety concerns or if continued participation does not appear in the best interest of the participant. At each trial site, all participants will be provided with standard of care throughout follow-up according to the national guidelines for antenatal care for India and Tanzania. Standard of care provided by clinic staff in Tanzania includes provision of daily iron-folic acid (IFA) supplements containing 60 mg iron and 0.4 mg folic acid. In India, pregnant women receive daily supplements containing 60 mg of iron throughout pregnancy and pregnant women less than 14 weeks of gestation are initially given daily 5 mg of folic acid until the end of the first trimester followed by daily 0.4 mg folic acid until delivery. Participants will have access to study clinics for post-trial care through the routine health system. In both trials, pregnant women who consent for enrollment will be individually randomized in a 1:1 ratio to either the (i) low-dose 500 mg calcium supplementation group or (ii) the standard high-dose 1500 mg calcium supplementation. The allocation sequence was generated by a statistician in Boston for each trial separately (India and Tanzania) using a computer-generated randomization list of 11,000 individuals with block randomization, stratified by study clinic. Two non-study staff hold the randomization list codes until completion of primary analyses or until requested by the Data and Safety Monitoring Board (DSMB). An independent study pharmacist in each country will privately prepare the 500 mg and 1500 mg regimen boxes with participant IDs based on the randomization list. At the randomization visit, research staff will assign pregnant women who are eligible and consent for trial randomization to the next available participant ID which corresponds to a set of pre-labeled regimen boxes. The trial statistician and data analysts will also be blinded to treatment allocation for the primary statistical analysis. As a result, the trial is quadruple blind with all trial participants, research staff, and care providers, Investigators, outcome assessors, and trials statistician and data analysts being unable to determine the allocated trial arm for any trial participant or identify trial participants who are on the same trial regimen. Further, the randomization procedures ensure complete allocation concealment. The DSMB, Institutional Review Boards (IRBs), attending physicians, and Principal Investigators may request to unblind a participant(s) allocated trial group during the trial for safety reasons. The sample size for each trial will be 11,000 participants. Statistical power for the primary outcomes of preeclampsia and preterm birth was calculated using formulas for binomial outcomes in non-inferiority trials [15]. Power calculations assumed 1:1 randomization, a one-sided test with a type I error rate (α) of 0.05, and a 10% loss-to-follow-up rate (including pregnancy loss for preterm outcome). The non-inferiority margin for each primary outcome was selected by the trials’ Technical Advisory Group. The non-inferiority margin for preeclampsia was set at a relative risk of 1.54 for low-dose calcium as compared to the standard high-dose calcium based on the lower bound of the 90% confidence interval for the effect of standard high-dose calcium versus placebo on preeclampsia [9, 16]. The non-inferiority margin for preterm birth was set at a relative risk of 1.16 for low-dose calcium as compared to the standard high-dose calcium regimen to maintain 50% of the effect based on the point estimate of the relative risk for the effect of standard high-dose calcium versus placebo on preterm birth (relative risk = 1.32) [9, 16]. The exact cumulative incidence of preeclampsia and proportion of preterm birth in the standard high-dose calcium arm was not known for the India and Tanzania sites, and therefore, power calculations were presented for a plausible range of cumulative incidence of preeclampsia of 1.5 to 5% and a proportion of preterm birth of 10.0 to 13.5%. Table ​Table11 presents statistical power in each trial for the plausible range of preeclampsia and preterm birth in the standard high-dose calcium group. For preeclampsia, each trial will have ~ 95% power if the cumulative incidence of preeclampsia is as low as 1.5% in the standard high-dose group and > 99.9% if the incidence is high at 5%. For preterm birth, each trial will have 84.3% power if the cumulative incidence is as low as 10% in the standard high-dose group and > 99.9% if the incidence is as high as 13.5%. Statistical power for primary outcomes of preeclampsia and preterm birth for each trial site based on a one-sided test with a type I error rate of 0.05 (India = 11,000 participants; Tanzania 11,000 participants) The schedule of trial enrollment, interventions, and assessments is presented in Fig. ​Fig.2.2. Participant study visits will include a randomization visit, pregnancy study visits every 4 weeks until delivery, a delivery visit, and a postnatal visit scheduled after 42 days postpartum (discharge). Mothers who have a fetal loss or a newborn death will be followed up after 42 days to ascertain maternal mortality. Participant retention will be promoted at clinic visits and through phone calls and home visits. Schedule of enrollment, interventions, and assessments (SPIRIT figure) All pregnant women will have a research visit at the time of randomization and every 4 weeks during pregnancy until the time of delivery. Starting at 32 weeks gestation, all women will also receive weekly phone calls and, if possible, will be asked to call the study team around the time of labor. All women will receive an ultrasound for gestational dating around the time of the randomization visit or shortly thereafter. In India, the ultrasounds will be performed by trained and authorized radiologists/sonographers using the ultrasound machines available at each facility. For quality control, Dr. Babu Phillips of the St. John’s Medical College Hospital will review images for each sonographer at the study sites every 6 months. In Tanzania, trained nurses will conduct ultrasounds with General Electronic Vscan Access portable ultrasound machines. All ultrasound images for the Tanzania site will be electronically stored and a random 10% for the duration of the trial will be assessed for quality by Dr. Blair Wylie and Ms. Adair Kendall (sonographer) of the Beth Israel Deaconess Medical Center in Boston and feedback will be given to sonographers in Tanzania for quality improvement. At all prenatal visits, study physicians will perform a full clinical examination, and treat all comorbidities in accordance with standard of care. Blood pressure will be assessed at randomization and at each pregnancy study visit. Electronic blood pressure monitors will be used in India (Omron HEM-7130, Kyoto, Japan) and Tanzania (Omron HEM-7131, Kyoto, Japan). Blood pressure assessment will be standardized by having all participants rest in a seated position for 5 min, placing the cuff on the left arm at the level of the heart and taking two blood pressure measurements (systolic and diastolic) at a 30-s interval. The electronic blood pressure machines will be calibrated once a month against a mercury sphygmomanometer; the electronic blood pressure machine will be discontinued from use in the study if the measured difference from the mercury sphygmomanometer is greater than 5 mmHg for systolic or diastolic blood pressure. Participants will be asked by study staff to provide urine at the randomization and each pregnancy follow-up visits, and the presence of protein will be assessed with a dipstick. Research staff will collect maternal height with calibrated stadiometers to the nearest 0.1 cm at the randomization visit. At randomization and all follow-up visits, maternal weight will be taken on digital weighing scales to the nearest 0.01 kg and the mid-upper arm circumference (MUAC) will be taken with a measuring tape to the nearest 0.1 cm. The weighing scales will be calibrated each day using certified 5, 10, and 20 kg weights. Pregnant women will have a finger-prick blood sample collected at randomization and at 32 weeks gestation for assessment of hemoglobin concentration (HemoCue Hb 301 in India and HemoCue Hb 201 in Tanzania, HemoCue AB, Ängelholm, Sweden). Hemocue machines will be calibrated each day using the tri-level control solutions provided by the manufacturer. Maternal dietary intake will be assessed with an open-ended 24-h diet recall conducted once during each trimester of pregnancy. Maternal depression will be assessed with the Patient Health Questionnaire (PHQ)-9 at randomization and at the 6 week postpartum visit [17]. We will also seek consent to collect 8 ml of venous blood specimen at randomization and 32 weeks gestation from participants enrolled at each site starting in January 2021 to understand the pathophysiology of hypertensive diseases of pregnancy and preterm birth using a nested case-cohort study design. Blood specimen will be collected in two vacutainers (4 ml each) with the anticoagulant ethylenediamine tetraacetic acid (EDTA) and temporarily stored in a cool box with icepacks until they are transported to a central laboratory at each site for processing within 4 h of collection in order to maintain the cold chain. At the laboratory, trained study technicians will centrifuge the blood specimen tubes to separate the plasma, which will then be aliquoted into multiple, prelabelled cryovials for long-term storage at − 80 °C freezers. Serum samples will be tested for biomarkers of calcium metabolism, pro- and anti-angiogenic profile, and other potential biomarkers related to preeclampsia and preterm birth. The selection of biomarkers and laboratory methods will be finalized in collaboration with the trials’ Technical Advisory Group in mid-2022 and will account for emerging evidence on the pathophysiology of preeclampsia and preterm birth. Study participants who consent for blood collection will also be asked for consent for long-term storage of serum samples and dried blood spots and their use in ancillary studies. Study staff will be available throughout the day and night to attend labor and delivery of participants. Participants who deliver outside of the study area will be reached by phone by the research staff to obtain relevant information from the mother and/or facility records. Clinic records and interviews with the clinic staff and participants will be used to ascertain the duration of each stage of labor, labor complications, and Apgar scores at 1 and 5 min for the newborns. Maternal blood pressure and proteinuria will be assessed by clinic or study staff or collected from clinic records. Infant length and weight will also be taken by study staff or recorded from facility records. Infant weight will be measured to the nearest gram using a digital scale and length with the use of a rigid length board with an adjustable foot piece to 1-mm precision. Verbal autopsies will be conducted to ascertain causes of maternal deaths [18]. Women or women/infant pairs will have a study visit at 6 weeks postpartum (42 days) and will be discharged from the trial after completion of this visit. At the postpartum visit, research staff will assess maternal and infant morbidity and hospitalization history. Nurses will also collect weight and, blood pressure from mothers. Study nurses will assess infant feeding, infant weight with a digital infant balance scale to the nearest gram and length to 1-mm precision with a rigid length board with an adjustable foot piece. Study nurses will measure infant head circumference and mid-upper arm circumference (MUAC) with flexible measuring tape. All infant anthropometric measurements will be recorded in duplicate. Verbal autopsies will be conducted to ascertain causes of infant deaths [18]. All data will be entered into independent electronic data capture systems in Tanzania and India, and the program will have in-built skip patterns and range check validations for each variable. All identifiable electronic data will remain in India and Tanzania and will be stored on secure local servers that are only accessible by the respective study data teams and Investigators.