One in seven babies in Africa are born with low birthweight, either because they are born too small (small-for-gestational age; SGA) or born too soon (preterm). Babies born SGA or preterm have a higher risk of dying, and do not grow as well in early life, leading to a higher risk of malnutrition. The most common form of malnutrition (stunting) affects health into adulthood, reduces learning at school, and lowers earning potential. To reduce child deaths and improve health, growth and prosperity throughout life, it is essential to improve birthweight. To do this, we need to understand the processes during pregnancy that lead to SGA and preterm. The fetus depends on a delicate balance of processes to grow properly and be delivered on time. Infections during pregnancy, and activation of the body's defence system to tackle infections (called inflammation) may disturb these processes, leading to SGA and preterm birth. Mothers with sexually transmitted infections, urinary tract infections, gum disease or diarrhoea during pregnancy have a higher risk of SGA and preterm birth. Inflammation can also occur even when a pregnant woman is not sick with an infection. There are trillions of bacteria in the body, called the microbiome, which generally do not cause disease. However, a change in composition of the microbiome can cause inflammation. Unbalanced vaginal, oral and gut microbiomes in pregnant mothers have all been associated with SGA and preterm birth, although most of these studies have been done in high-income countries. It is unclear whether and how disturbed microbiomes cause inflammation, SGA and preterm birth in Africa. We believe that taking antibiotics in pregnancy could improve birth outcomes by reducing harmful infections and inflammation. In order to test this, we will take advantage of an existing trial examining whether a daily antibiotic (called cotrimoxazole) during pregnancy can increase birthweight. We previously found that this antibiotic reduces inflammation as well as preventing infections. During the new study, 1000 women in rural Zimbabwe will receive either cotrimoxazole or placebo from the time they first book their pregnancy at the local clinic up until birth. The treatment will be decided randomly, like the flip of a coin. During pregnancy, oral samples (tongue swabs, dental plaque, saliva), vaginal swabs, stool, urine and blood will be collected from all mothers when they book and at 26, 34 and 36 weeks into pregnancy. Our first aim is to see whether infections and inflammation during pregnancy are associated with SGA and preterm birth. Women will be examined by a dentist to check their oral health and be tested for sexually transmitted infections, urine infections and diarrhoea, with treatment provided if needed. Using the oral, vaginal and stool samples collected from booking and the end of pregnancy, we will study the microbiome and inflammation to see if they are linked to SGA and preterm birth. Our second aim is to compare the microbiome and inflammation in 100 women receiving the antibiotic and 100 women receiving placebo. We will see whether cotrimoxazole reduces inflammation, infections, or changes the microbiome during pregnancy. We will also test whether bacteria become resistant to the antibiotic. Our third aim is to see what effect maternal antibiotics have on the baby. We will collect samples of the placenta, blood and stool at birth, and collect stool and blood again when they are 4 weeks old, to compare infections, microbiome and inflammation in babies whose mothers received antibiotics or placebo in pregnancy. Through this project, we hope to understand whether infections, microbiome disturbances and inflammation during pregnancy cause SGA and preterm birth, and exactly how the antibiotic works (if at all). This is important, because we may be able to design new treatments that can be given during pregnancy to help babies grow better and be born on time.

Our overarching hypothesis is that infections and inflammation during pregnancy drive small-for-gestational age (SGA) and preterm birth, and that cotrimoxazole treatment during pregnancy will ameliorate microbial and inflammatory exposures to promote healthy birth and growth. Our objectives and accompanying hypotheses in this proposal are: Objective 1: Undertake a longitudinal assessment of the vaginal, oral and intestinal microbiome, local and systemic inflammation during pregnancy to identify microbial and inflammatory exposures associated with SGA and preterm birth Hypothesis 1: Dysbiosis of the vaginal, oral and intestinal microbiomes during pregnancy is associated with local and systemic inflammation, which drive SGA and preterm birth. Objective 2: Compare vaginal, oral and intestinal microbiomes and inflammation between mothers randomised to receive daily cotrimoxazole or placebo during pregnancy Hypothesis 2: Cotrimoxazole in pregnancy reduces maternal pathogen carriage, modulates multi-site microbiomes and lowers local and systemic inflammation. Objective 3: Compare growth, hospitalisation, microbiomes, inflammation and anti-pathogen immune function between infants born to mothers randomised to cotrimoxazole or placebo. Hypothesis 3: Maternal cotrimoxazole reduces vertical pathogen transmission, promotes establishment of a healthier early-life microbiome, and suppresses placental and infant inflammation. To achieve these objectives, we have set the following milestones: 1. Revise trial protocol and statistical analysis plan 2. Secure ethics approvals and study sponsorship 3. Recruit and train research nurses, district dentist and trial obstetrician 4. Enrol 1000 pregnant women to a randomised controlled trial of cotrimoxazole versus placebo during pregnancy 5. Undertake baseline clinical assessments of pregnant women and longitudinal sample collection at baseline, 26, 34 and 36 gestational weeks 6. Monitor birth outcomes and undertake anthropometry at birth to identify SGA and preterm 7. Standardise laboratory methods in Zvitambo laboratory and finalise SOPs 8. Undertake neonatal sampling at delivery (meconium, placenta and cord blood) and 4 weeks post-partum (stool and blood) 9. Conduct metagenomic sequencing and analysis of maternal site-specific microbiomes (vaginal, oral and intestinal) and infant microbiomes (meconium and stool) 10. Conduct immunological assays of maternal site-specific inflammation (vaginal, oral, intestinal and systemic), placental inflammation, infant inflammation (stool and blood) and infant anti-pathogen immune cell function (blood) 11. Undertake hypothesis testing for objectives 1-3 using data generated from metagenomic, immunological and clinical assessments 12. Disseminate study findings to the community, Zimbabwean Ministry of Health, local policymakers and the local and international research community through community engagement, stakeholder meetings, conference presentations and research publications