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IntroductionThe use of low-dose aspirin by pregnant women to prevent preterm pre-eclampsia is gradually increasing. The administration of aspirin during pregnancy improves perinatal outcome, which could translate into improved child outcome in the long term. However, antenatal exposure to aspirin could have adverse effects on child development that may manifest later in life. The aim of this follow-up study is to assess the long-term effects of antenatal exposure to low-dose aspirin compared with placebo on survival, (neuro)development, behaviour and general health at 4 years corrected age.Methods and analysisThis is a follow-up study of the Dutch double-blind randomised controlled APRIL trial which assessed the effectiveness of treatment with aspirin (80 mg daily) compared with placebo for the prevention of preterm birth in women with a previous spontaneous preterm birth. Treatment was initiated before 16 weeks of gestation and continued until 36 weeks or birth. We aim to follow-up all 379 children born to women who participated in the APRIL trial and survived the neonatal period, at the corrected age of 4 years. The main outcomes are (neuro)development as assessed by the Ages and Stages Questionnaire, and behaviour as assessed by the Strength and Difficulties Questionnaire. Additional outcomes include mortality, growth and general health from birth up to 4 years, and a composite outcome including mortality, abnormal (neuro)development and problem behaviour. Analyses will be performed by intention-to-treat using a superiority design.Ethics and disseminationInstitutional Review Board approval was obtained from the Medical Research Ethics Committee from Amsterdam Medical Center (no. W20 289#20.325). The results will be published in a peer-reviewed journal and presented at conferences.Trial registration numberThe APRIL trial (NTR5675, NL5553; EudraCT number 2015-003220-31) and the APRIL follow-up study (NL8950) are registered in the Dutch trial register. The study is funded by the Amsterdam Reproduction & Development research institute.

Additional file 2: Figure S2. Median Fluorescence Intensity (MFI) per bacterium. The MFI of immunoglobulins bound to bacteria was measured during menstrual bleeding; time point 1 (T = 1), 7-11 days after onset of menstrual bleeding; time point 2 (T = 2) and 17-25 days after onset of menstrual bleeding; time point 3 (T = 3) and divided by the percentage of bacteria with bound IgA or IgG. Red line represents the median.

Additional file 9. Supplemental material 1. Validation of our protocol and gating strategies.

Additional file 8: Figure S8. The ratio between bound and unbound immunoglobulins in women with L. crispatus dominated and non-L. crispatus dominated microbiota over time. Bound and unbound immunoglobulins were measured menstrual bleeding; time point 1 (T = 1), 7-11 days after onset of menstrual bleeding; time point 2 (T = 2) and 17-25 days after onset of menstrual bleeding; time point 3 (T = 3) and divided to calculate the ratio. These ratios were compared between women with L. crispatus dominated vaginal microbiota and women having non-L. crispatus dominated vaginal microbiota over time. Red line represents the mean.

Additional file 4: Figure S4. The ratio between bound and unbound immunoglobulins. Bound and unbound immunoglobulins were measured during menstrual bleeding; time point 1 (T = 1), 7-11 days after onset of menstrual bleeding; time point 2 (T = 2) and 17-25 days after onset of menstrual bleeding; time point 3 (T = 3). Bound immunoglobulins (coating index) were divided by unbound immunoglobulins (concentration) to calculate the ratio. Red line represents the mean.

Additional file 7: Figure S7. Immunoglobulin levels in vaginal fluid from women with L. crispatus dominated and non-L. crispatus dominated microbiota over time. Unbound IgA, IgG, IgA1, IgA2 and SIgA in women with L. crispatus dominated vaginal microbiota compared to women having non-L. crispatus dominated vaginal microbiota over time. Data visualized as 10log of the unbound immunoglobulin concentration corrected for total protein. Red line represents the mean.

Additional file 6: Figure S6. Bacteria with bound IgG, IgG bound to bacteria and IgG Median Fluorescence Intensity (MFI) per bacterium in women with L. crispatus dominated and non-L. crispatus dominated microbiota over time. The (A) percentage of bacteria with bound IgG, (B) coating index of IgG bound to bacteria and (C) IgG MFI per bacterium in women with L. crispatus dominated vaginal microbiota compared to women having non-L. crispatus dominated vaginal microbiota over time. Red line represents the mean.

Additional file 1: Figure S1. Correlation between bacteria bound IgA and IgG. Correlation between bacteria bound IgA and IgG during menstrual bleeding; time point 1 (T = 1), 7-11 days after onset of menstrual bleeding; time point 2 (T = 2) and 17-25 days after onset of menstrual bleeding; time point 3 (T = 3). Data visualized as 10log coating index (CI, coating index = percentage of immunoglobulin bound bacteria * median fluorescence intensity).

Additional file 5: Figure S5. Microbiota composition over time. Heat map depicting the top 20 most abundant species among the 25 study participants during menstrual bleeding (1), time point 2 (2) and time point 3 (3). Colors reflect the relative abundance.

Additional file 3: Figure S3. Immunoglobulin levels in vaginal fluid over time. Unbound immunoglobulins were measured menstrual bleeding; time point 1 (T = 1), 7-11 days after onset of menstrual bleeding; time point 2 (T = 2) and 17-25 days after onset of menstrual bleeding; time point 3 (T = 3). The level of (A) unbound IgA1, (B) unbound IgA2 and (C) unbound SIgA over time. All data visualized as 10log of the unbound immunoglobulin concentration corrected for total protein. Red line represents the mean. * p < 0.05 ** p < 0.01.