L-ArGinine to pRevent advErse prEgnancy Outcomes (AGREE) (AGREE)

Oral Antenatal L-citrulline Supplementation to Reduce Adverse Pregnancy Outcomes: a Two-arm, Randomized, Placebo-controlled Multi-site Trial in Kenya

There are few safe, effective, and affordable interventions to improve pregnancy outcomes in low resource settings where the highest rates of poor birth outcomes occur. L-citrulline is naturally found in many foods and is changed into another important amino acid, L-arginine, in the body. L-arginine is important for the growth of a healthy placenta and healthy baby. Adding L-citrulline to the diets of pregnant women may be an effective and affordable way to improve the health of their babies.The goal of the AGREE trial is to test whether a dietary supplement containing a common food component, an amino acid called L-citrulline, can help pregnant Kenyan women at risk of malaria have healthier pregnancies and healthier babies. 2,960 pregnant Kenyan women will be enrolled and randomly assigned to take either a twice daily dietary supplement containing L-citrulline or a placebo supplement without additional L-citrulline. Maternal participants will be seen every month until delivery and at weeks 1 and 6 after birth. Infants will also be followed up at ages 6, 12, 18, and 24 months. The primary outcome of the study is 'adverse pregnancy outcome', a composite of foetal loss (miscarriage or still birth), preterm birth, low birth weight, small for gestational age or neonatal mortality. The results of the AGREE trial could help to guide obstetric and public health policy and provide a sustainable solution that could be implemented at the community level.

Study Overview

• Status: Not yet recruiting
• Conditions: Pregnancy; Preterm Birth; Malaria; Nutrition; Fetal Growth Restriction; Placental Development
• Intervention / Treatment: Dietary supplement: L-citrulline

Detailed Description

L-arginine is an essential amino acid in pregnancy and a key mediator of placental development and function. In many low-resource settings, widespread protein undernutrition contributes to L-arginine deficiency in pregnancy which is associated with an increased risk of adverse pregnancy outcomes. Using a preclinical model, we have previously shown that dietary L-arginine supplementation enhances placental vascular development and improves pregnancy outcomes. L-citrulline is an amino acid that is efficiently converted to L-arginine in the body and has a more palatable flavour profile. The primary objective is to to determine if daily antenatal oral supplementation with L-citrulline can reduce adverse pregnancy outcomes (defined as a composite of fetal loss, infants born preterm, small for gestational age or with low birthweight) among pregnant women at high risk of malaria and protein undernutrition in Kenya.This is an individually randomized, two-arm, parallel-group, placebo-controlled clinical trial involving 2,960 pregnant women randomly assigned to one of two study arms. The intervention arm will contain L-citrulline arm -twice daily 6.0 g sachet, each containing 5.00 g of quality-assured L-citrulline powder, 0.66 g maltodextrin and 0.30 g lactose anhydrous, 0.03 g citric acid, 0.01 g lemon flavour + antenatal standard of care with enhanced monitoring (n=1,480); or placebo arm containing 6.0 g sachet of quality-assured placebo, each consisting of 3.6 g maltodextrin and 2.4 g lactose monohydrate, 0.03 g citric acid, 0.01 g lemon flavour + antenatal standard of care with enhanced monitoring (n=1,480). All participants will continue to take the assigned product for 6 weeks after delivery and will receive an enhanced antenatal standard of care. The primary outcome is the clinical composite 'adverse pregnancy outcome'. Secondary outcomes include longitudinal assessments of physiological and molecular markers of endothelial function, angiogenesis, inflammation, placental function, L-arginine metabolism, neonatal sepsis, mortality, and early childhood neurocognitive development to age 24 months. The effect of L-citrulline supplementation on the composition of the participants' vaginal microbiota and the intestinal microbiota of both the participants and their newborns will be analysed in a subset of 132 mother/infant dyads. All maternal participants of the AGREE trial will be followed for 6 weeks post-partum and the children will be followed until age 2 years. Written informed consent will be obtained.

• Study Type: Interventional
• Enrollment (Estimated): 2960
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Feiko O. ter Kuile, PhD; Phone Number: +441517053287; Email: feiko.terkuile@lstmed.ac.uk
• Study Contact Backup: Name: Hellen C. Barsosio, MD; Phone Number: +254724464507; Email: hbarsosio@kemri.go.ke

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• Pregnant women aged 16-40 years,
• inclusive to 24 weeks gestational age as confirmed by ultrasound,
• who have a viable singleton pregnancy,
• are residents of the study area,
• willing to adhere to scheduled and unscheduled study visit procedures,
• willing to deliver in a study clinic or hospital

Exclusion Criteria:

• multiple pregnancies (i.e. twin/triplets);
• pre-existing hypertension, renal disease and/or diabetes, or severe anaemia (Hb < 5 g/dL);
• HIV-positive or HIV status unknown;
• malformations or nonviable pregnancy observed on enrolment ultrasound;
• known allergy or contraindication to any of the study supplements including lactose intolerance or observing a lactose-free diet;
• unable to give consent; or concurrent participation in any other clinical trial

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Quadruple

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: L-citrulline arm; L-citrulline arm is the intervention arm consisting of a twice daily 6.0 g sachet, each containing 5.000 g of quality-assured L-citrulline powder, 0.672 g maltodextrin and 0.286 g lactose anhydrous, 0.03 g citric acid, 0.012 g lemon flavour + antenatal standard of care with enhanced monitoring. The sachets will be provided at enrolment and each subsequent monthly ANC visit.	Dietary supplement: L-citrulline; Twice daily 6.0 g sachet, each containing 5.00 g of quality-assured L-citrulline powder, 0.66 g maltodextrin and 0.30 g lactose anhydrous, 0.03 g citric acid, 0.01 g lemon flavour + antenatal standard of care with enhanced monitoring; Other Names: L-arginine
No Intervention: Placebo arm; Placebo arm is the control arm consisting of a twice daily 6.0 g sachet of quality-assured placebo, each consisting of 3.6 g maltodextrin and 2.358 g lactose monohydrate, 0.03 g citric acid, 0.012 g lemon flavour + antenatal standard of care with enhanced monitoring. The sachets will be provided at enrolment and each subsequent monthly ANC visit.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Adverse pregnancy outcome	The primary outcome is 'adverse pregnancy outcome' defined as a composite of fetal loss (spontaneous abortion or stillbirth), singleton live births born SGA or with LBW, or preterm birth (PTB). 'Small for gestational age' will be defined using the INTERGROWTH population reference's 10th percentile. Fetal loss will be assessed monthly at scheduled ANC visits.	27 months

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Gestational hypertension	Assessed with systolic and diastolic blood pressure	27 months
Malaria infection during pregnancy	detected by microscopy and PCR (not for point of care) on peripheral blood	27 months
Placental malaria	detected by microscopy, by molecular methods, or by histology (past and active infection) on placental samples	27 months
Individual components of the placental malaria composite	detected by microscopy, by molecular methods, or by histology (past and active infection) on placental samples	27 months
Uncomplicated clinical malaria during pregnancy	RDTs will be used at the point of care for any patient presenting with fever, history of fever within 48h, or any other symptoms of clinical malaria infection. RDT-positivity is defined as either pLDH or HRP2 antigen positivity.	27 months
SARS-CoV-2 infection during pregnancy	Plasma samples will also be assayed for SARS-CoV-2 antibodies using validated techniques available at the time of analysis. If women are symptomatic a rapid SARS-COV-2 antigen test will also be conducted. If the rapid SARS-COV-2 antigen test is negative a confirmatory PCR will be conducted.	27 months
Maternal anaemia during pregnancy and delivery	Maternal anaemia is defined as haemoglobin concentration (Hb)<11g/dL; moderate maternal anaemia: Hb<9g/dL); severe anaemia: Hb<7g/dL); congenital anaemia: newborn Hb<12.5 g/dL.	27 months
Individual components of the adverse pregnancy outcome composite, and sub-composites	Including fetal loss (spontaneous abortions and stillbirth) and adverse livebirth (SGA-LBW-PTB composite). Gestational age will be assessed using ultrasound dating at enrolment. Preterm birth is defined as <37 weeks' gestation. Newborns will be weighed within 24 hours of delivery using digital scales (± 10 g) with LBW defined as <2,500g. Small for gestational age (SGA) will be defined as birth weight below the tenth percentile for a given gestational age and sex using the new INTERGROWTH reference population. Neonatal length and stunting will be assessed within 24 hours of delivery. Infants will be defined as stunted if their height-for-age is more than two standard deviations below the WHO Child Growth Standards median.	27 months
Fetal growth	estimated by validated ultrasound and maternal biomarkers	27 months
Birthweight-for-gestational age	Gestational age will be assessed using ultrasound dating at enrolment. Small for gestational age (SGA) will be defined as birth weight below the tenth percentile for a given gestational age and sex using the new INTERGROWTH reference population.	27 months
Neonatal length and stunting	Newborns will be measured for length within 24 hours of delivery. Infants will be defined as stunted if their height-for-age is more than two standard deviations below the WHO Child Growth Standards median.	27 months
Congenital anaemia	congenital anaemia: newborn Hb<12.5 g/dL.	27 months
Congenital malaria infection	detected by microscopy and PCR (not for point of care) on cord blood samples	27 months
Congenital SARS-CoV-2 infection	SARS-CoV-2 antibodies detected on cord blood samples	27 months
Neonatal death	vital status on discharge (alive/dead), vital status at 7 days (alive/dead) and 28 days (alive /dead) post admission will be documented.	27 months
Perinatal mortality	vital status on discharge (alive/dead), vital status at 7 days (alive/dead)	27 months
Composite of fetal loss and neonatal mortality	miscarriage, still births or vital status on discharge (alive/dead), vital status at 7 days (alive/dead) and 28 days (alive /dead) post admission will be documented.	27 months
Neonatal sepsis	WHO Integrated Management of Childhood Illness criteria128, specifically any one of the following signs (i) not able to feed at all or not feeding well, (ii) convulsions, (iii) severe chest indrawing, (iv) high body temperature (380C or above), (iv) low body temperature (less than 35.50C), (v) movement only when stimulated or no movement at all (vi) in infants less than 7 days old, fast breathing (60 breaths per minute or more).	27 months
Early childhood neurocognitive development	Early childhood neurocognitive development will be assessed longitudinally over the first two years of life using a combination of questionnaires, direct assessments, and objective measures appropriate to the developmental periods within this time frame. The Home Observation for Measurement of the Environment (HOME) is a 58-question assessment. The WHO Motor Development Milestones checklist is a simple, WHO-validated assessment of six gross motor milestones in early childhood development. The Mullen Scales of Early Learning (MSEL) is a comprehensive evaluation assessing early childhood development in five domains. The MacArthur Bates Communication Developmental Inventory (MCAB-CDI)132 is an interview-style questionnaire that consists of 100 vocabulary items, 6 gesture items, and 5 grammatical items to assess communication/language development.	27 months
Allergic reaction	defined as anaphylaxis, hives/rash after taking the supplement.	27 months
Maternal mortality	Maternal mortality will be defined as the death of a woman while pregnant or within 42 days of termination of pregnancy, irrespective of the duration and site of the pregnancy, from any cause related to or aggravated by the pregnancy or its management but not from accidental or incidental causes. A verbal autopsy questionnaire will attempt to determine the cause of death.	27 months
Congenital abnormalities	any abnormality detected in surface and clinical examination at birth and week 1 and 6-8	27 months
Vomiting study supplement	vomiting within 30 minutes of taking the supplement	27 months
Gastrointestinal complaints	including nausea, dyspepsia, diarrhea reported at scheduled and unscheduled visits and and through follow-up phone calls and home visits	27 months
Symptoms of dizziness or syncope or palpitations	Study staff will administer a questionnaire to assess for the occurrence of tolerability adverse events (including nausea, dyspepsia, diarrhoea, dizziness, palpitations) at scheduled and unscheduled visits, and and through follow-up phone calls and home visits	27 months
Markers of L-arginine bioavailability and nitric oxide biogenesis	L-arginine bioavailability will be assessed by plasma concentrations of L-arginine, ADMA and the L-arginine/ADMA ratio. Plasma SDMA will also be quantified.	27 months
Markers of endothelial function, placental function and inflammation	including plasma concentrations of Angiopoietin (Ang)-1, Ang-2, soluble Tyrosine kinase with immunoglobulin-like and EGF-like domains (sTIE)1, sTIE2, Vascular Endothelial Growth Factor (VEGF), soluble VEGF-receptor1, soluble Endoglin (sEng), Placental Growth Factor (PLGF), soluble Intercellular Adhesion Molecule (sICAM), soluble Tumour Necrosis Factor (sTNF) receptor 2 (sTNFR2), C5a, Chitinase-3-like protein 1 (CHI3L1), C-reactive protein (CRP), Interleukin (IL)-18 binding protein (IL-18BP), IL-6, Pregnancy-associated Protein A (PAPP-A), beta-human chorionic gonadotropin (β-hCG); and urine concentrations of protein and complement	27 months
Evidence of malaria or SARS-CoV-2 vertical transmission	Laboratory and nutritional outcomes	27 months
Evidence of SARS-CoV-2 infection	Laboratory and nutritional outcomes: (antigen, PCR, and/or serology)	27 months
Mediators of host immune function	Concentrations of circulating mediators of host immune function, response, endothelial function, and nutrition in the newborn at birth and six weeks of life	27 months
Microbial diversity	(N=132 maternal and N=132 newborn participants). Shannon diversity and other measures of microbial diversity richness and abundance in maternal intestinal and vaginal microbiota at enrolment and the first post-treatment timepoint, across gestation and at six weeks post-partum, and in newborn intestinal microbiota at six weeks of life. Nutritional and microbial composition of breast milk	27 months

Collaborators and Investigators

• Sponsor: Liverpool School of Tropical Medicine
• Collaborators: University of Toronto; Kenya Medical Research Institute; Telethon Kids Institute
• Investigators: Principal Investigator: Kevin Kain, PhD, University of Toronto; Study Director: Julie Wright, MD, University of Toronto

Study record dates

Study Major Dates

• Study Start (Estimated): September 30, 2023
• Primary Completion (Estimated): December 30, 2025
• Study Completion (Estimated): December 30, 2026

Study Registration Dates

• First Submitted: April 5, 2023
• First Submitted That Met QC Criteria: June 28, 2023
• First Posted (Actual): July 6, 2023

Study Record Updates

• Last Update Posted (Actual): July 6, 2023
• Last Update Submitted That Met QC Criteria: June 28, 2023
• Last Verified: June 1, 2023

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Pathologic Processes; Fetal Diseases; Pregnancy Complications; Obstetric Labor Complications; Obstetric Labor, Premature; Growth Disorders; Female Urogenital Diseases and Pregnancy Complications; Urogenital Diseases; Premature Birth; Fetal Growth Retardation
• Other Study ID Numbers: 19-109; PACTR202303697293140 (Registry Identifier: The Pan African Clinical Trials Registry (PACTR))

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: Biological samples and data will be shared using material and data transfer agreements with the collaborating institutions to minimise the risk of unauthorised analysis beyond the scope of the agreed parameters. The full protocol will be available on request to any interested professional and may be published in peer-reviewed journals or deposited in an online repository. Individual, de-identified participant data will be made available for meta-analyses as soon as the data analysis is completed, with the understanding that results of the meta-analysis will not be published prior to the results of the individual trial without the prior agreement of the investigators. A fully de-identified data set of the complete patient-level data will be available for sharing purposes, such as via WWARN repository platform http://www.wwarn.org/working-together/sharing-data/accessing-data).
• IPD Sharing Time Frame: No later than five years after the publication of the trial.
• IPD Sharing Access Criteria: Collaborating institutions. All requests for data for secondary analysis will be considered by the Data Access Committee to ensure that the use of data is within the terms of consent and ethics approved.
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP; ICF; ANALYTIC_CODE; CSR

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No