Multi-centered, parallel groups, controlled, triple blind, randomized clinical trial (RCT) subdivided in 2 strata as a function of the hemoglobin (Hb) levels at the start of the pregnancy:
Stratum #1: If the levels of Hb are situated between 110 and 130 g/L, the individual will be randomly assigned to an iron dose supplement of 40 or 80 mg/d
Stratum #2: If the levels of Hb are >130 g/L, the individual will be randomly assigned to an iron dose supplement of 40 or 20 mg/d
The overall design of the study is summarized in Figure 1. This trial has been registered with EU Clinical Trials Register, EUCTR-2012-005480-28.
Methods: participants, interventions, and outcomes
The study will be conducted in 10 Primary Care Centers (PCC) of the Catalunya Sexual and Reproductive Health-care Service [Atención a la Salud Sexual y Reproductiva (ASSIR)] of the Catalan Institute of Health [Instituto Catalán de la Salud (ICS)]. These Centers provide care for 3,500 potential pregnancies per year. The specialist health-care workers include gynecologists and midwives. The participating reproductive health-care services (RHS) provide cover for urban, suburban, and rural PCCs.
The participants will need to fulfill the following inclusion, and none of the exclusion, criteria.
Inclusion criteria: Adult female older than 18 years of age with ≤ 12 weeks gestation without anemia (Hb >110 g/L), capable of understanding the official State languages (Castilian or Catalan), who can understand the characteristics of the study, and who sign the informed consent form.
Exclusion criteria: Multiple pregnancy, taking >10 mg iron during the months prior to week 12, hypersensitivity to egg protein (due to the iron prescription formula contains ovalbumin), previous serious illness (immunosuppressed status) or chronic illness that could affect the nutritional development (e.g. cancer, diabetes), malabsorption, and liver disease such as chronic hepatitis or cirrhosis. The risk factors of pregnancy to be considered as exclusion criteria are: adverse obstetric history, previous uterine surgery, heart disease grade 2, 3 or 4, endocrinopathy, suspected fetal malformation, morbid obesity, preeclampsia, maternal infection, uterine malformation, perinatal recurrent death, alcoholism, diabetes 1 and 2, uterine cervical incompetence.
The clinical follow-up of the pregnancy in the PCC will be according to the program set by RHS. This includes a clinical visit at recruitment into the present study, a visit every trimester, and one at 40 days post-partum (Figure 1).
In the recruitment visit before week 12 of the pregnancy, the inclusion criteria will be assessed (except the Hb levels and the number of fetuses) as well as the exclusion criteria. Informed consent will be solicited. A clinical history will be recorded, as will a questionnaire regarding the ingestion of iron supplements, multi-vitamins, or other treatments (Drugs_Q) and, if a smoker, the Fagerstrom test for tobacco dependency. A blood sample for standard biochemical analyses (including hemoglobin) will be sent for processing in the centralized laboratory.
At visit 1, around the 12th week of gestation, Hb levels will be evaluated as will be the number of fetuses (using echography) to confirm that the inclusion criteria are fulfilled. If fulfilled, the individuals will be retained in the study and, if not, will be transferred out of the study, and considered a screening failure. The remaining women will be assigned to Stratum #1 or Stratum #2 of the study and will be randomized with respect to iron supplement prescription. Clinical history will be taken, including the use of multi-vitamins and iron supplements (on the questionnaire abbreviated as “Drugs_Q”) and the following questionnaires filled-in: Food Frequency Questionnaire (FFQ); International Physical Activity Questionnaire (IPAQ); State-Trait of Anxiety Inventory (STAI) and the Fagerstrom questionnaire (Fagerstrom_Q). The ultrasound data on the fetus will be recorded. Venous blood will be taken for analyses, the results of which will be reviewed in the next clinical visit. The iron supplementation that will be needed at the next visit will be prepared for distribution. Adverse events occurring since the previous visit will be recorded.
At visit 2, around week 24 of gestation, clinical history will be taken, and will include the Drugs_Q, which, from this visit onwards, includes the adherence to the iron supplementation prescribed. The following questionnaires are filled-in: FFQ; IPAQ; STAI, Fagerstrom_Q. The fetal ultrasound data will be registered. The biochemical analyses/results will be reviewed and a further blood sample taken for analysis, the results of which will be reviewed at the next clinical visit. The iron supplementation that will be needed at the next visit will be prepared for distribution. Adverse events occurring since the previous visit will be recorded.
At visit 3, around week 36 of gestation, the clinical history will be taken, the Drugs_Q as well as the FFQ; IPAQ; STAI, Fagerstrom_Q questionnaires will be filled-in. The fetal ultrasound data will be recorded and the biochemical results will be evaluated. A further blood sample will be taken for analyses, the results of which will be discussed at the next clinical visit.
The iron supplementation that will be needed at the next visit will be prepared for distribution. Adverse events occurring since the previous visit will be recorded.
At visit 4 (40 days post-partum), the clinical history will be taken, the Drugs_Q and the FFQ; IPAQ; STAI; Fagerstrom_Q questionnaires will be filled-in. A questionnaire on post-partum depression (Edinburgh Postnatal Depression Scale; EPDS) and the Parenting Stress Index (PSI) will be applied. The standard laboratory analyses results will be discussed. A further blood sample will be taken for analyses. Data on birth and the newborn will be recorded (weight and height). Clinical history of the baby will be recorded, including: anthropometric data (weight, height, head circumference) and cognitive development will be assessed (Bayley III), as well as behavioral and temperament (Early Infancy Temperament Questionnaire; EITQ). Adverse events occurring since the previous visit will be recorded.
Starting from around the 12th week of gestation and continuing up to partum, adherence to prescription will be recorded. The dose assignments will be random and followed-up blind for each RCT stratum. The principal active ingredient is ferrimanitol ovoalbumin. The doses of 20 mg, 40 mg and 80 mg per day of elemental iron correspond to 150 mg, 300 mg and 600 mg ferrimanitol ovoalbumin. The reference dose is 40 mg/d, similar to that routinely prescribed in clinical practice .
Biological samples of the RCT
The blood extractions at visits 1, 3 and at birth, will be more than are normally taken for biological analyses in a RCT. This is because slightly larger amounts are required for subsequent analyses such as specialized biochemical measurements (ferritin, cortisol) and genetics (C282Y and H63D of the HFE gene). These samples will be treated and stored for subsequent analyses in the BioBank of the reference hospital of the participating PCCs.
Participants will be free to withdraw from the trial at any time, and without the need to provide any reason. The information that may already have been collected on participants will still be used. Withdrawal from the study will not affect the standard of care received from the practice. If participants withdraw before randomization, they will be replaced by an appropriate substitute.
The pregnancies diagnosed as anemic during the RCT will have the iron supplementation from the RCT changed, and will be treated according to the standard clinical practice protocol. These women will continue forming part of the study and will have the same follow-up, except they will be considered as therapeutic failures in the intent-to-treat statistical analyses, and will not be taken into account in the protocol analysis.
This visit will take place only in case of the participant’s withdrawal from the study at any time outside the scheduled timetable. The exact date and reason for discontinuation will be registered. If possible, the healthcare professional will assess the woman’s health status via clinical history notes, and an ultrasound. A blood sample will be solicited for routine tests. The adherence to the iron supplementation up until the discontinuation of the treatment will be recorded, as will any adverse events. All the questionnaires will be filled-in, including: FFQ; IPAQ; STAI, Fagerstrom_Q. In case of having given birth, all the variables related to delivery and the newborn will be collected and a questionnaire on post-partum depression (Edinburgh Postnatal Depression Scale; EPDS) and the Parenting Stress Index (PSI) will be applied.
Principal variables in the pregnancy are iron status of the mother at each clinical visit (anemia, ferropenic anemia, risk of hemoconcentration) and birth weight of the baby.
Definition of main outcome variables
Anemia is defined as Hb <110 g/L in the 1st and 3rd trimester, Hb <105 in 2nd trimester, Hb <120 g/L post-partum .
Ferropenic anemia is defined as: Hb < the normal limit, and serum ferritin (SF) <15 μg/L .
Hemoconcentration risk is defined as: Hb >130 g/L in the 2nd and 3rd trimester .
Clinical follow-up of the pregnancy in the PCC will be according to the program set by ASSIR. The participant timeline is detailed in Figure 1
Recruitment visit (previous to week 12 of gestation)
1st visit of the study (around 12th gestational week)
2nd visit of the study (around 24th gestational week)
3rd visit of the study (around 36th gestational week)
4th visit of the study (40 days post-partum)
The principal variables taken into account in calculating the size of sample needed are iron deficiency anemia, risk of hemoconcentration in the third trimester of pregnancy, and birth weight of the baby.
To achieve the study’s main objective, sample size is calculated in accordance with the following parameters: an alpha risk of 0.05 and a beta risk of 0.20 in a one-tailed test of comparison. A drop-out rate of 35% is factored-in.
To calculate the sample size needed to reduce the prevalence of iron deficiency anemia and hemocentration risk by 10 points, we consulted previous data from our research group [5, 6, 8]. We had observed a prevalence of 23.5% of iron deficiency anemia in the 3rd trimester in pregnant women with Hb levels of 110-130 g/L in the first trimester and a prevalence of risk of hemoconcentration of 14.7% in the 3rd trimester of pregnant women who started pregnancy with Hb levels of 130–150 g/L.
We found that, in Stratum #1 of the RCT, it will be necessary to include 283 women in each group and in Stratum #2 of the RCT 156 women would be necessary in each group.
With respect to child development, to detect differences in weight of the newborn ≥200 g taking into account a standard deviation (SD) of 450 g , the estimation suggests that it will be necessary to have 106 women in each group.
Hence, the overall study sample is calculated as 878 women: 566 in Stratum #1 and 312 in Stratum #2.
Methods: intervention assignment
The pregnant women are assigned to Stratum #1 or Stratum #2 as a function of the hemoglobin values in the baseline analysis of the study. They are, then, randomly assigned to 2 treatment groups to receive different iron supplements. The randomization is performed using centralized computer software, which is automatic and masked and applies to the electronic data collection forms, as well. The procedure for randomization is independent for each Stratum.
The study will be triple blind: the participant, the health-care professional, and the statistician. The treatment drug will be administered “blind” i.e. the doses are not identifiable since the packaging has the same format, presentation, and visual characteristics.
For Stratum #1, the treatments will be designated as A or B, and for Stratum #2 they will be designated as C or D.
The laboratory of MEIJI TEDEC FARMA, SA will be responsible for manufacturing, packaging and labeling the study medications.
Only MEIJI TEDEC FARMA, SA and the Clinical Pharmacology Service of the Vall d’Hebron Hospital in Barcelona will know the distribution codes and the composition of each of the treatments.
There would be no need for un-blinding except if an unexpected serious adverse event occurs. In which case, the pharmaco-vigilance staff of TEDEC-MEIJI FARMA S.A. will take responsibility for un-blinding and communicating the adverse event to the appropriate health authorities. TEDEC-Meiji Farma SA will not reveal the treatment codes until the end of the trial, when these data and the documents generated will be made available to the Principal Investigator (VA) and the Promoter (Jordi Gol i Gurina).
Methods: data collection, management and analyses
Data collection methods
Midwifes will be responsible for data collection from the clinical history, and from the questionnaires. They will introduce the data into an electronic data collection format which will be monitored by an external service.
The ultrasound measurements will be conducted by two obstetricians using previously-standardized techniques.
Clinical history of the mother: date of birth, socio-economic status, parity, date of last menstruation, corrected date of last menstruation, estimated date of partum, risk factors during pregnancy, pregnancy planning, previous use of contraceptives, clinical antecedents, surgery and personal obstetric data, toxic habits, blood pressure, height, weight of the mother (self-reported at the recruitment visit and measured objectively at each clinical follow-up visit). Similar data from the father will be solicited.
Questionnaire on iron supplement intake and of multi-vitamins, adherence to iron supplementation and possible adverse effects and concomitant treatments (Drugs_Q)
Food Frequency Questionnaire (FFQ) validated by our research team 
International Physical Activity Questionnaire (IPAQ) [24, 25]
State-Trait Anxiety Inventory (STAI) 
Fagerstrom test to evaluate tobacco dependency (Fagerstrom_Q) 
Edinburgh Postnatal Depression Scale (EPDS) [28, 29]
Parenting Stress Index (PSI) [30, 31]
Standard laboratory analyses: Hemogram: hemoglobin, mean corpuscular volume, hematocrit (Coulter, Hialeah, FL, USA)
Biological samples for the measurement of biochemical parameters (ferritin, cortisol) and genetic polymorphisms (C282Y and H63D of the HFE gene)
Ultrasound #1: confirmation of the date of the last menstruation and date of gestation corrected for echography, prenatal diagnostics, chorion characteristics, amniotic fluid and determination of secondary markers
Ultrasound #2: biometry, morphological evaluation, gender, position and presentation of the fetus, body and cardiac activity, characteristics of the placenta and umbilical cord, and status of the fetus
Ultrasound #3: evaluation of fetal growth using biometry, body and cardiac activity, characteristics of the placenta, of the umbilical cord, and position and status of the fetus
Clinical history of the newborn (gender, status of the newborn, weight and height at birth) and anthropometric measurements of weight, height, head circumference at 40 days
Bayley III scales: indices to evaluate mental and psychomotor development 
Early Infancy Temperament Questionnaire (EITQ) : test to evaluate behavior and temperament
Procedures for extraction, transfer and storage of biological samples
Blood samples will be collected in 2 tubes; 1 tube of 7.5 ml containing EDTA as anticoagulant and 1 tube of 7.5 ml without anticoagulant. The samples will be transported to the BioBank for immediate analyses to generate the hematology profile using the laboratory’s Coulter analyzer. In the BioBank, the samples will be prepared for storage. Before processing, the samples in the EDTA tubes will be inversion-mixed 10 times to ensure that the blood is well mixed before centrifugation at 4°C to separate plasma which will be stored in aliquots (500 μl) at -80°C for measurements described later. DNA will be extracted and stored at -80°C for subsequent genetic analyses. The tube without anticoagulant will be left without mixing for 30 minutes at room temperature to enable coagulation. The serum will be separated by centrifugation, distributed in aliquots of 500 μl and stored at -80°C.
The stored samples in the BioBank will be thawed at the end of the clinical study and processed simultaneously to minimize inter-batch variation:
Serum ferritin (immunochemiluminescence)
C-reactive protein (immunoturbidimetry)
Genetic analyses (only in the first blood sample): mutation C282Y, H63D of the HFE gene (PCR and digestion with specific enzymes)
To control for possible bias, the intra- and inter-assay coefficients of variation will be calculated for all the measured variables. Values recommended by the “kit” manufacturers are accepted. All the biochemical determinations of all the samples in a series will be performed by the same person so as to minimize inter-assay variations in the longitudinal measurements of variables that are of interest to the investigators.
The description of the variables studied will be performed using conventional techniques. Variables with non-normal distribution will be transformed as necessary for normalization of distribution of values. The Kolmogorov-Smirnov and the Shapiro-Wilks test will be used to verify normality of distributions.
Analysis of the primary outcome
The effects of iron dose supplement in each RCT on the biochemical iron status and mother-child health will be compared using regression models adjusted for those variables that can influence the relationship. Logistic regression or Cox models will be applied for qualitative variables such as, for example, the percentage of anemia or hemoconcentration at the end of pregnancy. Linear multiple regression models will be applied for dependent quantitative variables. The models will be adjusted for those variables that biologically affect the relationships studied, such as the serum ferritin levels, presence of alterations in the HFE gene, age of the mother, gestational age, parity, anthropometric indices, diet, and lifestyle, and the interactions between these variables. Initially included in the model will be all those variables that form part of the theoretical model and, in a second phase, the variables for entry into the model will be selected step by step (forward and backward) to achieve the most reduced stable models.
Conditions for the application of models will be verified using standard techniques that are based, essentially, on residuals analysis. The bilateral null hypothesis of normality, no difference, and non-significance of the regression coefficients, will be rejected when their probabilities are <5%. The data will be analyzed using the latest version of SPSS/PC package for Windows.
Data will be analyzed in 2 ways:
a) Intention-to-treat analysis
The principle of “intention-to-treat” is a way of analyzing the results that considers all individuals entered into the study, according to the group to which they were originally assigned, although they may not have complied with the protocol. In this case, in all the circumstances that have led to not having hemoglobin determinations before delivery, we will proceed to incorporate at this time-point the immediately-prior determination of hemoglobin. All randomized participants randomized who had received at least one dose of study medication will be included.
b) Per-protocol analysis
Included in the analysis will be only those pregnant women who received the allocated intervention at randomization. The per-protocol population (PP) will include those participants who had taken at least the 80% of the study medication, with proper assessment of adherence, or without any major protocol violations.
Pregnant women diagnosed with anemia at the start will be treated according to the usual protocol, and not as a part of the current study. Women diagnosed with anemia posteriorly, will be treated with standard therapy, but will remain part of the study in the intention-to-treat analysis. However, they will not be considered in the per-protocol analysis.
Data missing from the study variables will be treated with the direct likelihood method  when no data is available. In these cases, the profile of pregnant women will be used to adjust the estimates of these parameters.
An analysis of sensitivity will be performed using a mixed model based on constraint-based patterns of missing values without dependence on the future to assess the robustness of the main results of the Mixed Model Repeated Measures against the possible transgression of random default assumption.
To ensure correct conduct and security of the RCT according to the requirements of good clinical practice, external services will be contracted to perform the tasks of monitoring of the participating centers according to the requirements of the Spanish Agency of Medicines and Health Products [Agencia Española de Medicamentos y Productos Sanitarios; AEMPS].
All adverse events suffered will be recorded at each clinical visit and assessed for severity, expectedness and causality.
Ethics and dissemination
Research ethics approval
In Spain, approval was obtained from the Clinical Research Ethics Committee of the Jordi Gol Research Institute in Primary Care [Instituto de Investigación en Atención Primaria; IDIAP] and from the Spanish Agency for Medicines and Health Products [Agencia Española del Medicamento y Productos Sanitarios; AEMPS].
The study is designed according to the requirements of the Declarations of Helsinki, and the Tokyo update.
All participants are informed of the study, its objectives and activities related to their participation: number and schedule of visits, diagnostic tests, results information. Patients will be provided with detailed information leaflets, and will be asked to sign a consent form.
Confidentially will be guaranteed for all participants in the study. The study conforms to the Government Data Protection Act for Personal Information (15/1999 of 13th Dec. LOPD).
Access to data
All documents will be stored securely and accessible only to trial staff and authorized personnel.
All scientific publications must have the approval of the principal investigator (VA).
The principal investigator of the study is committed to making public such results as become available, both positive and negative. When studies and research are made public to the scientific community, the funding sources and the laboratory TEDEC-MEIJI FARMA S.A will be acknowledged.
The researchers are committed to making raw, anonymity-guaranteed, data sets available to the scientific community upon legitimate request to the principal investigator, once the trial is completed.