Effectiveness of Adaptation of the Dose of Iron Supplementation in Pregnancy on Maternal-child Health. (ECLIPSES)

The study will be conducted in 2 Primary Care Centers (PCC) from Tarragona and Reus of the Catalunya Sexual and Reproductive Healthcare Service [Atención a la Salud Sexual y Reproductiva (ASSIR)] of the Catalan Institute of Health [Instituto Catalán de la Salud (ICS)]. 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 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.

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, which include date of birth, socioeconomic 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.

Moreover, a questionnaire regarding the ingestion of iron supplements, multi-vitamins, or other treatments 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 and the questionnaires filled-in about food consumption, physical activity, anxiety status and tobacco dependency. ). A physical examination will be performed to measure weight and blood pressure. The ultrasound data on the fetus will be recorded to assess Crown Rump Length (CRL). A physical examination will be performed to measure weight and blood pressure. 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 questionnaire about use of multi-vitamins and iron supplements which, from this visit onwards, includes the adherence to the iron supplementation prescribed. The questionnaires about food consumption, physical activity, anxiety status and tobacco dependency are filled-in. A physical examination will be performed to measure weight and blood pressure. The fetal ultrasound data will be registered to assess status of fetus and estimated fetal weight. 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 questionnaire about use of multi-vitamins and iron supplements as well as the questionnaires about food consumption, physical activity, anxiety status and tobacco dependency will be filled-in. A physical examination will be performed to measure weight and blood pressure. The fetal ultrasound data will be recorded to assess status of fetus and estimated fetal weight 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 questionnaire about use of multi-vitamins and iron supplements as well as the questionnaires about food consumption, physical activity, anxiety status and tobacco dependency will be filled-in. A questionnaire on post-partum depression and the Parenting Stress Index will be applied. The standard laboratory analyses results will be discussed. A further blood sample will be taken for analyses. Data on birth (type of delivery) and the newborn will be recorded (weight and height). Clinical history of the baby will be recorded, including: gender, status of newborn, Apgar score, anthropometric data (weight, height, head circumference), breastfeeding and levels of vitamin D. Cognitive development will be assessed, as well as behavioral and temperament. Adverse events occurring since the previous visit will be recorded.

Sample size:

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 two tailed test of comparison. A drop-out rate or lack of data of 35% is factored-in.

To calculate the sample size, previous data from the research group of investigators were consulted (Aranda, 2011, Hernández-Martínez, 2011, Ribot, 2012). A prevalence of 23.5% of iron deficiency anemia was observed 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.

• In Stratum 1, to reduce the frequency of anemia ferropenic from 23.5% to 11.5% in the intervention group supplemented with 80mg/day of iron with respect to the group supplemented with 40mg/day, will be necessary to include 236 women in each group
• In Stratum 2, to reduce the frequency of hemocontration from 14.7% to 2.7% in the intervention group supplemented with 20mg/day of iron with respect to the group supplemented with 40mg/day, will be necessary to include 116 women in each group

Intervention assignment: Allocation

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.

Stratum 1: If Hb from 110 to 130 g/L, randomly assigned at week 12 to receive iron supplement of 40 or 80 mg/d.

Stratum 2: If Hb >130 g/L, randomly assigned at week 12 to receive iron supplement of 40 or 20 mg/d.

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.

Blinding 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. 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 (Institut d'investigació en Atenció Primària, IDIAP, Jordi Gol i Gurina ).

Statistical methods 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

Monitoring 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].