Gates Open Res Gates Open Research 2572-4754 F1000 Research Limited London, UK 10.12688/gatesopenres.15120.1 Study Protocol Articles Prevalence and determinants of anaemia during the second or third trimester of pregnancy in Bangladesh: a cross-sectional study protocol

[version 1; peer review: 1 approved, 1 approved with reservations]

 Ahmed Shamim Methodology Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-3369-2379 1 Hasan Mohammed Imrul Funding Acquisition Methodology Writing – Original Draft Preparation Writing – Review & Editing 1 2 Rahman A M Quaiyum Project Administration Writing – Review & Editing 1 Bhuiyan Mohammad Saiful Alam Project Administration Writing – Review & Editing 1 Tipu S M Mulk Uddin Project Administration Writing – Review & Editing 1 Braat Sabine Formal Analysis Writing – Review & Editing https://orcid.org/0000-0003-1997-3999 3 McLean Alistair R D Formal Analysis Writing – Review & Editing 2 3 Arifeen Shams El Funding Acquisition Writing – Review & Editing 1 Hamadani Jena D Funding Acquisition Methodology Writing – Review & Editing 1 Pasricha Sant-Rayn Conceptualization Funding Acquisition Methodology Writing – Review & Editing 2 4 5 Davidson Eliza M Methodology Writing – Review & Editing a 2 Maternal and Child Health Division, icddr,b, Dhaka, GPO Box 128, 1000, Bangladesh Population Health and Immunity Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia Centre for Epidemiology and Biostatistics, School of Population and Global Health, The University of Melbourne, Melbourne, Victoria, 3050, Australia Diagnostic Haematology and Clinical Haematology, The Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia Diagnostic Haematology and Clinical Haematology, Peter MacCallum Cancer Centre, Parkville, Melbourne, Victoria, 3050, Australia davidson.e@wehi.edu.au

*Contributed as first author

Competing interests: S-RP reports consultancy fees from CSL-Vifor Pharma, ITL-Biomedical and Atomo Diagnostica. He is Director of the WHO Collaborating Centre for Anaemia Detection and Control (unfunded). No other competing interests were disclosed.

 8 3 2024 2024 8 23 29 2 2024 Copyright: © 2024 Ahmed S et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Anaemia in pregnancy remains a critical global health problem and has long-term adverse health outcomes for both the mother and child. In developing countries like Bangladesh, there has been limited progress in reducing the burden of anaemia in pregnancy. This community-based survey will provide insight into the prevalence and determinants of anaemia during the second and third trimesters of pregnancy.

 Methods

This cross-sectional survey will be conducted in three Upazilas (sub-districts) in rural Bangladesh. A total of 1,500 pregnant women will be recruited in second and third trimester, calculated from last menstrual period. The primary outcome is to determine the prevalence of anaemia (Hb<11 g/dL); haemoglobin will be measured in venous blood by HemoCue. The association between iron deficiency and anaemia will be determined, and the role of drinking water iron level in anaemia aetiology will be explored. Key secondary outcomes include the prevalence of moderate (Hb <10 g/dL) and severe anaemia (Hb <7 g/dL), iron deficiency (ferritin <15 mg/L), and iron deficiency anaemia (concurrent iron deficiency and anaemia). Detailed information on socioeconomic status, current and past pregnancies, knowledge on nutrition and anaemia, water-sanitation-hygiene and maternal mental health status will be collected.

 Conclusions

This study will provide comprehensive information on the prevalence and determinants of anaemia in second and third trimester of pregnancy, and inform anaemia intervention strategies to improve maternal and child health outcomes.

 Ethics and dissemination

This study has been approved by the Ethical Review Committee of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) and the Human Research Ethics Committee of the Walter and Eliza Hall Institute of Medical Research (WEHI), Melbourne, Australia. The results will be actively disseminated through reports and presentations to stakeholders, symposiums and scientific publications.

 Registration

The protocol is registered with the Australian and New Zealand Clinical Trials Registry ( ACTRN12621000982819; 26/07/2021).

 Anaemia iron deficiency pregnancy gestational age Bangladesh Gates Foundation GR-02015 INV-017316 Australian National Health and Medical Research Council GNT2009047 Australian National Health and Medical Research Council GNT1158696 This work was supported by the Gates Foundation (INV-017316; GR-02015). S-RP is funded by the Australian National Health and Medical Research Council (NHMRC) Fellowships (GNT1158696 and GNT2009047). The funder has no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Anaemia in pregnancy causes a range of adverse effects and remains a critical global health problem 1 . In 2019, 37% of all pregnancies, including 47% in Southern Asia, were affected by anaemia 2 . Worldwide, compilations of the prevalence of anaemia show a large variation among regions, with prevalence highest in low and middle-income countries (LMICs) 3 . There has been limited progress in reducing the burden of anaemia among pregnant women in many LMICs.

Reflecting this challenge and the global attention on anaemia, reduction in the prevalence of anaemia by 50% among women of reproductive age is now (as of 2020) a Sustainable Development Goal indicator as well as a key World Health Organization (WHO) Global Nutrition target 4, 5 . WHO estimates that approximately 50% of anaemia in pregnancy worldwide, and 47% of anaemia in pregnancy in South Asia, would be responsive to iron interventions 1 .

In Bangladesh, anaemia is considered a major public health problem 6, 7 , yet, there is a paucity of data on the prevalence of anaemia in pregnancy in Bangladesh. Globally, the predominant cause of anaemia is iron deficiency 8– 10 . However, the contribution of iron deficiency to anaemia in Bangladesh remains largely unknown. This is further complicated by areas of high iron in groundwater 11 , which is used as the main drinking water source in Bangladesh 12 . One study found that women of reproductive age in a rural area of northern Bangladesh had no iron deficiency, which was attributed to high drinking groundwater iron 13 . Overall, it remains unclear whether iron in drinking water is protective against iron deficiency in Bangladesh.

We will conduct a cross-sectional survey of anaemia prevalence in pregnant women during the second and third trimester. We will also determine the prevalence of iron deficiency and anaemia, and explore the association between iron status, drinking water iron and anaemia This survey will determine the disease burden in the Narayanganj district of Bangladesh and enable further studies to design strategies for anaemia prevention in pregnancy.

 Methods Objectives and outcomes

The primary objective is to determine the prevalence of anaemia (haemoglobin, Hb <11 g/dL) during the second and third trimester (13–32 weeks) of pregnancy in women from Rupganj, Sonargaon and Bandar Upazilas (sub-districts) of Narayanganj district.

The key secondary objectives are to determine the prevalence of moderate anaemia (Hb <10 g/dL), severe anaemia (Hb <7 g/dL), iron deficiency (ferritin <15 mg/L), and iron deficiency anaemia (concurrent anaemia and iron deficiency). The association between the woman’s drinking water iron level and anaemia status will be assessed, along with the association between the woman’s iron status and anaemia status.

Secondary objectives are to describe women’s sociodemographic characteristics, pregnancy history, antenatal and delivery care seeking practices and challenges, nutritional knowledge and existing practices for anaemia management, delivery preparedness and autonomy, water-sanitation-hygiene practices, mental health status, and COVID-19 history.

Exploratory objectives include investigating the genetic, immune, metabolic and molecular aetiologies related to anaemia. These analyses are beyond the scope of this protocol and will be reported separately.

 Study design

This will be a cross-sectional survey to determine the prevalence of anaemia (Hb <11g/dL) in pregnant women during the second and third trimester (13–32 weeks).

 Eligibility criteria

Inclusion criteria

In their second or third trimester (13–32 weeks of gestation, dated by Last Menstrual Period).

Resident in the study catchment area.

Exclusion criteria

No consent.

 Study setting and participants

This will be conducted in three Upazilas (sub-districts) of the Narayanganj district: Rupganj, Sonargaon and Bandar. Each Upazila is subdivided into a number of unions. Each union is subdivided into nine wards. There are six or more Family Welfare Assistant (FWA) units in ward level depending upon the area and size of population. Each Upazila comprises of ~350,000–600,000 people, and estimated pregnancies is ranging from ~8,000 to ~12,000. The study area has low levels of iron in the groundwater 14, 15 . There is no data on the prevalence of anaemia in pregnant women from this district.

 Study procedures

Screening. All women of reproductive age living in the study site are screened regularly for pregnancy ( via questioning on last menstrual period (LMP)) by Family Welfare Assistants (FWAs), and pregnant women are identified and recorded in a FWA register. The FWAs work principally in FWA units in rural areas in the Upazila (sub-district) set up under Directorate General of Family Planning (DGFP). Study Field Research Assistants (FRAs) will collect the names with other details of the pregnant women from FWA register to prepare a pregnancy database. Calculated from first day of LMP, they will identify pregnant women in their second or third trimester (13–32 weeks) and arrange a household visit. When LMP dates are missing, FRAs will confirm the date during household visits. During the home visits, FRAs will verify that the women fulfil the eligibility criteria, ensuring that they are 13–32 weeks pregnant and a resident in the study catchment area. Following the acquisition of informed consent, the participants will be asked to provide information on their socio-demographics, household assets, income, smoking status, and autonomy.

Water iron testing

Drinking water iron level will be measured from freshly collected water from the family drinking water source. The HACH iron test kit, Model IR-18B will be used for iron testing.

Enrolment

Physical measures

Following FRAs recommended date, participants will attend a nearby Family Welfare Centre, and a physician will perform a physical examination ensuring privacy of the participants. This will include a general examination: checking for pallor, blood pressure, pulse, oedema, and anthropometric measurements: height, weight and mid-upper arm circumference.

Questionnaires

All participants will be asked to provide information pertaining to their current & previous pregnancy history, including anaemia testing & treatment, iron-folic acid (IFA) use, nutritional knowledge & practice, delivery preparedness & complication readiness, and COVID-19 history. A water, sanitation and hygiene (WASH) questionnaire will also be conducted 16 . Finally, women’s mental health will be measured using the Edinburgh Postnatal Depression Scale (EPDS) 17– 19 .

Blood collection & Haemoglobin (Hb) measurement

After all aseptic precautions, the study physician will collect 5 ml of venous blood sample. Immediately after collection of blood, Hb level will be measured from venous blood using HemoCue® Hb 301 portable haemoglobin analyzer (Hemocue, Inc., Ängelholm, Sweden).

Laboratory procedures

At the field site laboratory, lab technicians will do the initial sample processing. The field site lab will have small laboratory equipment for sample separation, and storage. Lab technicians will use standard operating procedures (SOPs) for all laboratory processing of samples, developed by a senior Laboratory Technologist. Samples will be stored initially at -20°C but periodically will be transferred for long-term storage to International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) lab where there is a -80°C freezer.

Blood samples: separation and long-term storage

Blood will be centrifuged, and serum will be separated from the blood cells as soon as possible. Blood will also be stored in appropriate preservatives to enable iron and inflammatory biomarker measurements (serum ferritin and CRP) and further analysis of genetic, immune, metabolic and molecular aetiologies related to anaemia. Residual whole blood (EDTA) samples from participants will be stored for future extraction of DNA and/or RNA.

 Assessment of safety

COVID-19 precautions. Considering the global pandemic of COVID-19, the icddr,b has its own guidelines and SOPs regarding infection prevention and control (IPC) to be implemented. All contacts with participants during data collection or physical measurements procedures will strictly follow IPC SOPs. For bio-sample collection and processing in the field and storage in the lab we will follow Bio-Safety Level (BSL) guidelines. The central biosafety team of icddr,b is in charge of the compliance management. All study staff will receive training on IPC and necessary personal protective equipment prior to commencement of field activities.

 Data handling and record keeping

Data will be collected using smart phones (7 inch) with internet enabled SIM cards for data collection and transmission from the community to a central database server at icddr,b. The data management system is designed to run on an android platform and configured based on the working areas of data collectors. Data will be stored locally in the device during interview, and uploaded to icddr,b servers on a regular basis. The databases will be password protected and only members of the study team and investigators will have access. Only deidentified data (study identification numbers but no names) will be stored on the database.

Database lock. We will declare and document the data as being clean only once the validation process is concluded. The database will be locked only after all decisions on the evaluability of the data from each individual subject have been made and documented.

 Statistical considerations

Sample size calculations. We will screen 1,500 pregnant women (500 in each Upazila (sub-district)). Overall, 40% will be recruited from each of the second and third trimesters respectively and the remaining 20% will be recruited on a competitive basis. With a minimum sample size of 600 women across all Upazilas (sub-districts) per trimester, the precision of the two-sided 95% confidence interval of the true underlying prevalence is at most ±4% (assuming prevalence of 50%) using the Wald method to measure the two-sided 95% confidence interval. The expected precision is ±3.8% assuming the prevalence of anaemia in pregnancy is (210/600) 35% ( i.e., 95% CI 31.2% to 38.8%), ±2.4% assuming the prevalence of moderate/severe anaemia in pregnancy is (60/600) 10% ( i.e., 95% CI 7.6% to 12.4%), and ±3.9% assuming the prevalence of iron deficiency in pregnancy is (240/600) 40% ( i.e., 95% CI 36.1% to 43.9%).

Data analysis. The proportion of women with anaemia, moderate anaemia, severe anaemia, iron deficiency, and iron deficiency anaemia will be obtained per trimester, along with two-sided 95% confidence intervals. Women’s sociodemographic characteristics, antenatal and delivery care seeking practices and challenges, nutritional knowledge and existing practices for anaemia management, preparedness and autonomy, water-sanitation-hygiene practices, mental health status, COVID-19 history, and laboratory outcomes will be summarised per trimester.

Univariable and multivariable regression models will be used to explore the association between potential determinants of anaemia and anaemia collected in the study. A causal diagram informed by clinical expertise will be developed to help determine which factors to include in the multivariable models. The association between the woman’s ferritin, iron deficiency status, haemoglobin, anaemia status and her family’s drinking water iron level will be explored similarly. We will present the estimate and two-sided 95% confidence interval of each association alongside two-sided p-values. Data will be analysed using STATA (StataCorp, Statistical Software: College Station, TX).

 Study oversight

Quality control and quality assurance. Authorised representatives of the sponsor, an Institutional Ethics Committee/Review Board (IRB) or regulatory authority may visit the study site to perform audits or inspections, including source data verification.

 Ethics and dissemination

Ethical considerations

Research ethics approval

This protocol, the informed consent forms and any subsequent modifications will be reviewed and approved by the Institutional Review Board (IRB) of icddr,b as well as by the human research ethics committee of WEHI, Melbourne, Australia. Approval from the ethics committee at icddr,b and WEHI was obtained on the 29 th of March, 2021 (PR-20125) and on the 2 nd of June, 2021 (21/5) subsequently prior to the commencement of the study.

Ethical review

The IRB of icddr,b will review the study activities anytime during the study period and will provide feedback.

Informed consent

Informed written consent will be obtained from each participant before conducting any study related procedure. The PI will ensure that the participant (and the subject's parent/guardian in case of assent) is given full and adequate oral and written information about the study. Information will be provided in the local language of the participant. The participant will be given the opportunity to ask questions and allowed time to consider the information provided.

 Risks to the study participants

Blood sampling. A total of 5 ml of blood will be collected through a venepuncture. A small bruise or mild pain on the venepuncture site from where the blood is taken may develop. All efforts will be made to minimise pain, discomfort, and fear for pregnant women. Only well-trained and fully qualified staff will be hired for this project. Only new disposable needles and lancets are used for the blood taking procedures, and these will be discarded immediately after their use.

 Subject data protection

Participant confidentiality is strictly held in trust by the participating investigators, research staff, and the sponsoring institution and their agents. This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to participating participants. The study protocol, documentation, data and all other information generated will be held in strict confidence. No information concerning the study, or the data, will be released to any unauthorised third party, without prior written approval of the sponsoring institution. All laboratory specimens, evaluation forms, reports and other records that leave the site will be identified only by the Participant’s Unique Identification Number to maintain participant confidentiality. Clinical information will not be released without written permission of the participant, except as necessary for monitoring by regulatory agencies.

 Study status

Participants (n=1500) have been screened and enrolled, and blood samples have been tested for anaemia status. Data is currently being analysed.

 Discussion

The prevalence of anaemia during pregnancy is a significant concern worldwide, with negative health consequences for both the mother and the child that can persist over time 20– 22 . This study aims to estimate the prevalence of anaemia during the second and third trimester of pregnancy. Additionally, this study aims to elucidate the contribution of drinking water iron and iron deficiency to anaemia in this setting. The findings of this study will be used to develop effective anaemia intervention strategies that can improve maternal and child health outcomes. The dissemination of the results will be done through various channels such as reports, presentations, symposiums, and scientific publications. This approach will ensure that the stakeholders are kept informed about the progress of the project and the outcomes. It will also enable the wider scientific community to access and benefit from the findings.

 Data availability Underlying data

No data are associated with this article.

 Acknowledgements

This research protocol was funded by Bill & Melinda Gates Foundation. Icddr,b acknowledges with gratitude the commitment of Bill & Melinda Gates Foundation to its research efforts. Icddr,b is also grateful to the Government of Bangladesh and Canada for providing core/unrestricted support.

 WHO: The global prevalence of anaemia in 2011.World Health Organization: Geneva (CH),2015;44. Reference Source World Health Organisation: Prevalence of anaemia in pregnant women (aged 15-49) in 2019. The Global Health Observatory.2023; (accessed 17 May 2023). Reference Source DeMaeyer E Adiels-Tegman M : The prevalence of anaemia in the world. World Health Stat Q. 1985;38(3):302–316. 3878044 Sustainable Development Solutions Network: Goal 02. End hunger, achieve food security and improved nutrition, and promote sustainable agriculture.(accessed Sep 18, 2023). Reference Source WHO/UNICEF: The extension of the 2025 maternal, infant and young child nutrition targets to 2030. June 13,2019; (accessed Sep 18, 2023). Reference Source Ahmed F : Anaemia in Bangladesh: a review of prevalence and aetiology. Public Health Nutrition. 2000;3(4):385–393. 11135792 10.1017/s1368980000000446 National Institute of Population Research and Training (NIPORT), Mitra and Associates, ICF International: Bangladesh Demographic and Health Survey 2011. Dhaka, Bangladesh and Calverton, Maryland, USA: NIPORT, Mitra and Associates, and ICF International.2013. Reference Source World Health Organization, United Nations Children’s Fund/United Nations University: Iron deficiency anaemia. Assessment, prevention and control. A guide for programme managers. Geneva: World Health Organization,2001. Reference Source Gillespie S Kevany J Mason J : Controlling iron deficiency. Nutrition Policy Discussion Paper. (UN/ACC),1991. Reference Source Stevens GA Finucane MM De-Regil LM : Global, regional, and national trends in haemoglobin concentration and prevalence of total and severe anaemia in children and pregnant and non-pregnant women for 1995-2011: a systematic analysis of population-representative data. Lancet Glob Health. 2013;1(1):e16–25. 25103581 10.1016/S2214-109X(13)70001-9 4547326 British Geological Survey & Department for Public Health Engineering GotPsRoB: Arsenic Contamination of Groundwater in Bangladesh.BGS Technical Report WC/00/19 [DG Kinniburgh and PL Smedley, editors],2001. National Institute of Population Research and Training (NIPORT), ICF: Bangladesh Demographic and Health Survey 2017-18. Dhaka, Bangladesh, and Rockville, Maryland, USA: NIPORT and ICF,2020. Reference Source Merrill RD Shamim AA Ali H : Iron status of women is associated with the iron concentration of potable groundwater in rural Bangladesh. J Nutr. 2011;141(5):944–949. 21451130 10.3945/jn.111.138628 Hasan MI Hossain SJ Braat S : Benefits and risks of Iron interventions in children (BRISC): protocol for a three-arm parallel-group randomised controlled field trial in Bangladesh. BMJ Open. 2017;7(11): e018325. 29146650 10.1136/bmjopen-2017-018325 5695407 Pasricha SR Hasan MI Braat S : Benefits and risks of iron interventions in infants in rural Bangladesh. N Engl J Med. 2021;385(11):982–995. 34496174 10.1056/NEJMoa2034187 Jolly SP Roy Chowdhury T Sarker TT : Water, sanitation and hygiene (WASH) practices and deworming improve nutritional status and anemia of unmarried adolescent girls in rural Bangladesh. J Health Popul Nutr. 2023;42(1): 127. 37957706 10.1186/s41043-023-00453-8 10644610 Gausia K Fisher C Algin S : Validation of the Bangla version of the Edinburgh Postnatal Depression Scale for a Bangladeshi sample. J Reprod Infant Psychol. 2007;25(4):308–315. 10.1080/02646830701644896 Gausia K Hamadani JD Islam MM : Bangla translation, adaptation and piloting of Edinburgh Postnatal Depression Scale. Bangladesh Med Res Counc Bull. 2007;33(3):81–7. 18783062 10.3329/bmrcb.v33i3.1138 Hamadani JD Tofail F Hilaly A : Association of postpartum maternal morbidities with children's mental, psychomotor and language development in rural Bangladesh. J Health Popul Nutr. 2012;30(2):193–204. 22838161 10.3329/jhpn.v30i2.11313 3397330 Balarajan Y Ramakrishnan U Ozaltin E : Anaemia in low-income and middle-income countries. Lancet. 2011;378(9809):2123–35. 21813172 10.1016/S0140-6736(10)62304-5 Daru J Zamora J Fernández-Félix BM : Risk of maternal mortality in women with severe anaemia during pregnancy and post partum: a multilevel analysis. Lancet Glob Health. 2018;6(5):e548–54. 29571592 10.1016/S2214-109X(18)30078-0 Haider BA Olofin I Wang M : Anaemia, prenatal iron use, and risk of adverse pregnancy outcomes: systematic review and meta-analysis. BMJ. 2013;346: f3443. 23794316 10.1136/bmj.f3443 3689887 10.21956/gatesopenres.16456.r38618 Reviewer response for version 1 Mahdy Zaleha Abdullah 1 Referee https://orcid.org/0000-0003-0856-0697 Department of Obstetrics and Gynaecology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia

Competing interests: No competing interests were disclosed.

 28 12 2024 Copyright: © 2024 Mahdy ZA 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The study protocol has been well thought out and relevant to the current global situation.  The authors have given an excellent background in relation to the choice of problem and setting.  Study design and methodology are appropriate.  Data collection tools and plans are detailed and systematic.  It would add value to the study if the outcome of the pregnancies recruited is followed up and documented, assuming that this is not difficult to do and bearing in mind that there might be some dropouts or incomplete data from this follow up exercise.  Looking at the dates, I assume that the data collection for this study has already been completed.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Yes

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

Maternal fetal medicine, particularly preeclampsia, preterm labour, maternal nutrition specifically anemia in pregnancy, healthcare technology innovation, and medical education with a focus on simulation based education and training.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.21956/gatesopenres.16456.r36187 Reviewer response for version 1 Lewkowitz Adam 1 Referee https://orcid.org/0000-0002-8706-1409 Department of Obstetrics and Gynecology, Warren Alpert Medical School, Brown University, Providence, Rhode Island, USA

Competing interests: No competing interests were disclosed.

 22 5 2024 Copyright: © 2024 Lewkowitz A 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

This excellently written study protocol provides clear insight into a high-quality, well-designed cross-sectional study that the authors have completed to identify the prevalence and determinants of anemia during the second or third trimester of pregnancy in Bangladesh. My main criticism was that this study protocol was published AFTER all the study data had been collected instead of before, but that is not uncommon and, perhaps some of the following comments would be of use for the manuscript (or can be utilized to further clarify this study protocol).

1) The introduction heavily emphasizes the high groundwater iron in Bangladesh but then in the study setting mentions that the specific Upazilas have low groundwater iron. Why emphasize this in the introduction, then?

2) How were the thresholds for anemia and ferritin chosen for this study?

3) Did any of the participants receive obstetric ultrasounds for confirmator dating?

4) The EPDS has been validated in Bangladesh, as is evident by the citations. Perhaps mention this in the text, however

5) Are there population/demographic differences in the Upazilas? Will the authors conduct subgroup analyses per Upazila?

6) What does recruitment on a competitive basis mean?

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Yes

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

Iron deficiency anemia in pregnancy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.