NOTE

Primary results blinding by scrambling treatment arm. Some patterns in blinded results due to correlations between HM components

Treatment arms

Misame-3: Examine the effects of fortified food supplements during pregnancy and/or lactation on milk composition. Intervention arms: * (Control) Prenatal iron/folic acid (IFA) maternal supplementation and postnatal IFA maternal supplementation * Prenatal balanced energy-protein (BEP) maternal supplementation and postnatal IFA maternal supplementation * Prenatal IFA maternal supplementation and postnatal BEP maternal supplementation * Prenatal BEP maternal supplementation and postnatal BEP acid maternal supplementation

Vital: Analyze the impact of fortified food supplements during lactation and the administration of azithromycin prophylaxis to infants on human milk. Intervention arms: * Control * BEP+Ex.BreastFeed * BEP+Ex.BreastFeed+azithromycin

Elicit: Evaluate how nicotinamide administration to mothers and infants, as well as antimicrobial prophylaxis to infants, influences milk composition. Intervention arms: * (Control) Placebo to mothers and infants * Nicotinamide to mothers and infants, placebo to infants * Placebo to mothers and infants, Azithromycin & Nitazoxanide to infants * Nicotinamide to mothers and infants, Azithromycin & Nitazoxanide to infants

Outcomes

  • Primary: Macro- and micro-nutrients
  • Secondary: HMO’s, targeted proteins/bioactives
  • Tertiary: Targeted Metabolomics
  • Exploratory: Untargeted metabolomics, untargeted proteomics, microbiome

Statistical Analysis

Primary

  • Covariate adjusted TMLE analysis, fit using SL ensemble machine learning
  • Analyzing timepoints separately
    • Include HM component changes as outcomes
  • Analyze intervention impacts on both the raw concentration and Z-scored outcomes
  • Multiple comparison correction: We will correct for multiple comparisons by using FDR correction on the P-value for overall intervention effects on each outcome (chi-sq test).
  • Missing data imputation-
    • Outcomes: following lab recommendations using same cleaning process as for other papers (Nolan’s code)
    • Covariates: “Missing” level for categorical variables, median for continuous variables with missingness indicator added.

Secondary

  • Intervention effects on dimension-reduction scores by laboratory datasets (two approaches- PCA and SuperLearner weighted outcome score)
  • Subgroup analysis stratified by maternal BMI and season of milk collection

Sensitivity

  • Unadjusted (regression) analysis
  • Negative and positive control analyses

Tables 1-3, baseline covariates by arm

Note Not adjusting for maternal anthropometry measures (BMI, gestational weight gain)

Control
(N=224)		 BEP/BEP
(N=200)		 BEP/IFA
(N=165)		 IFA/BEP
(N=244)		 Overall
(N=833)
sex
Female 118 (52.7%) 86 (43.0%) 78 (47.3%) 139 (57.0%) 421 (50.5%)
Male 106 (47.3%) 114 (57.0%) 87 (52.7%) 105 (43.0%) 412 (49.5%)
mage
Mean (SD) 25.1 (5.35) 23.5 (5.57) 24.6 (5.57) 23.6 (5.83) 24.2 (5.62)
Median [Min, Max] 24.0 [17.0, 39.0] 22.5 [16.0, 36.0] 23.0 [16.0, 40.0] 22.5 [15.0, 39.0] 23.0 [15.0, 40.0]
meducyrs
Mean (SD) 2.77 (3.39) 2.32 (3.27) 2.95 (3.54) 3.92 (4.05) 3.03 (3.64)
Median [Min, Max] 2.00 [0, 10.0] 2.00 [0, 10.0] 2.00 [0, 10.0] 2.00 [0, 13.0] 2.00 [0, 13.0]
nrooms
Mean (SD) 2.41 (1.96) 2.49 (1.64) 2.48 (1.25) 2.39 (1.55) 2.44 (1.64)
Median [Min, Max] 2.00 [1.00, 12.0] 2.00 [1.00, 9.00] 2.00 [1.00, 6.00] 2.00 [1.00, 9.00] 2.00 [1.00, 12.0]
parity
Mean (SD) 2.46 (1.47) 2.41 (1.27) 2.58 (1.32) 2.33 (1.44) 2.43 (1.39)
Median [Min, Max] 2.00 [0, 8.00] 2.00 [0, 6.00] 2.00 [0, 7.00] 2.00 [0, 7.00] 2.00 [0, 8.00]
nperson
Mean (SD) 6.16 (4.12) 6.56 (5.08) 6.82 (4.07) 6.36 (5.09) 6.45 (4.65)
Median [Min, Max] 5.00 [2.00, 23.0] 5.00 [2.00, 27.0] 6.00 [2.00, 20.0] 5.00 [2.00, 30.0] 5.00 [2.00, 30.0]
imp_water_src
Improved 131 (58.5%) 108 (54.0%) 98 (59.4%) 157 (64.3%) 494 (59.3%)
Unimproved 93 (41.5%) 92 (46.0%) 67 (40.6%) 87 (35.7%) 339 (40.7%)
impfloor
0 94 (42.0%) 74 (37.0%) 66 (40.0%) 95 (38.9%) 329 (39.5%)
1 130 (58.0%) 126 (63.0%) 99 (60.0%) 149 (61.1%) 504 (60.5%)
cookplac
missing 224 (100%) 200 (100%) 165 (100%) 244 (100%) 833 (100%)
dvseason
Rainy Season 166 (74.1%) 168 (84.0%) 137 (83.0%) 188 (77.0%) 659 (79.1%)
Summer Season 58 (25.9%) 32 (16.0%) 28 (17.0%) 56 (23.0%) 174 (20.9%)
dlvloc
1 11 (4.9%) 3 (1.5%) 3 (1.8%) 6 (2.5%) 23 (2.8%)
3 204 (91.1%) 184 (92.0%) 153 (92.7%) 215 (88.1%) 756 (90.8%)
4 9 (4.0%) 13 (6.5%) 9 (5.5%) 23 (9.4%) 54 (6.5%)
hhwealth
Mean (SD) -0.0807 (2.49) 0.111 (2.33) 0.778 (2.61) -0.263 (2.59) 0.0823 (2.53)
Median [Min, Max] -0.217 [-5.62, 7.76] 0.351 [-4.38, 4.79] 1.06 [-5.06, 6.01] -0.624 [-5.10, 6.77] 0.0975 [-5.62, 7.76]
hhfoodsecure
Mean (SD) 3.17 (3.38) 3.15 (2.84) 2.97 (2.84) 3.86 (3.91) 3.33 (3.34)
Median [Min, Max] 2.00 [0, 14.0] 3.00 [0, 11.0] 3.00 [0, 12.0] 3.50 [0, 14.0] 3.00 [0, 14.0]
nperson_miss
Mean (SD) 0 (0) 0.0150 (0.122) 0 (0) 0 (0) 0.00360 (0.0599)
Median [Min, Max] 0 [0, 0] 0 [0, 1.00] 0 [0, 0] 0 [0, 0] 0 [0, 1.00]
parity_miss
Mean (SD) 0.201 (0.402) 0.280 (0.450) 0.291 (0.456) 0.250 (0.434) 0.252 (0.434)
Median [Min, Max] 0 [0, 1.00] 0 [0, 1.00] 0 [0, 1.00] 0 [0, 1.00] 0 [0, 1.00]
meducyrs_miss
Mean (SD) 0.138 (0.346) 0.190 (0.393) 0.0727 (0.260) 0.0984 (0.298) 0.126 (0.332)
Median [Min, Max] 0 [0, 1.00] 0 [0, 1.00] 0 [0, 1.00] 0 [0, 1.00] 0 [0, 1.00]
hhfoodsecure_miss
Mean (SD) 0 (0) 0.0150 (0.122) 0 (0) 0 (0) 0.00360 (0.0599)
Median [Min, Max] 0 [0, 0] 0 [0, 1.00] 0 [0, 0] 0 [0, 0] 0 [0, 1.00]
Control
(N=107)		 Az.
(N=88)		 Nico
(N=96)		 Nico+Az.
(N=106)		 Overall
(N=397)
sex
Female 52 (48.6%) 35 (39.8%) 56 (58.3%) 58 (54.7%) 201 (50.6%)
Male 55 (51.4%) 53 (60.2%) 40 (41.7%) 48 (45.3%) 196 (49.4%)
mage
Mean (SD) 30.9 (7.69) 28.3 (6.91) 28.4 (6.97) 29.5 (6.63) 29.3 (7.12)
Median [Min, Max] 32.0 [19.0, 42.0] 26.0 [18.0, 42.0] 27.0 [19.0, 46.0] 29.0 [18.0, 46.0] 28.0 [18.0, 46.0]
meducyrs
Mean (SD) 6.85 (2.59) 6.36 (2.63) 6.77 (2.42) 6.19 (2.97) 6.55 (2.67)
Median [Min, Max] 7.00 [0, 13.0] 7.00 [0, 12.0] 7.00 [0, 11.0] 7.00 [0, 13.0] 7.00 [0, 13.0]
nrooms
Mean (SD) 4.89 (2.17) 4.59 (2.27) 4.48 (2.12) 4.91 (2.24) 4.73 (2.20)
Median [Min, Max] 4.00 [1.00, 11.0] 4.00 [1.00, 12.0] 4.00 [2.00, 11.0] 5.00 [2.00, 12.0] 4.00 [1.00, 12.0]
parity
Mean (SD) 4.92 (2.82) 3.88 (2.47) 3.92 (2.55) 4.55 (2.57) 4.35 (2.64)
Median [Min, Max] 5.00 [1.00, 12.0] 3.00 [1.00, 12.0] 3.00 [1.00, 9.00] 5.00 [1.00, 12.0] 4.00 [1.00, 12.0]
nperson
Mean (SD) 7.54 (2.48) 7.14 (2.81) 6.81 (2.86) 7.45 (2.59) 7.25 (2.69)
Median [Min, Max] 7.00 [3.00, 14.0] 7.00 [3.00, 14.0] 7.00 [1.00, 18.0] 8.00 [3.00, 14.0] 7.00 [1.00, 18.0]
imp_water_src
Improved 73 (68.2%) 58 (65.9%) 64 (66.7%) 64 (60.4%) 259 (65.2%)
Unimproved 34 (31.8%) 30 (34.1%) 32 (33.3%) 42 (39.6%) 138 (34.8%)
impfloor
missing 107 (100%) 88 (100%) 96 (100%) 106 (100%) 397 (100%)
cookplac
No 35 (32.7%) 32 (36.4%) 42 (43.8%) 42 (39.6%) 151 (38.0%)
Yes 72 (67.3%) 56 (63.6%) 54 (56.3%) 64 (60.4%) 246 (62.0%)
dvseason
Post-Harvest 59 (55.1%) 59 (67.0%) 56 (58.3%) 66 (62.3%) 240 (60.5%)
Pre-Harvest 48 (44.9%) 29 (33.0%) 40 (41.7%) 40 (37.7%) 157 (39.5%)
dlvloc
1 40 (37.4%) 43 (48.9%) 44 (45.8%) 58 (54.7%) 185 (46.6%)
4 67 (62.6%) 45 (51.1%) 52 (54.2%) 48 (45.3%) 212 (53.4%)
hhwealth
Mean (SD) -0.261 (0.611) 0.182 (1.14) -0.0165 (1.07) 0.139 (1.09) 0.00300 (1.00)
Median [Min, Max] -0.360 [-0.807, 2.32] -0.0258 [-0.807, 5.66] -0.360 [-0.807, 5.66] -0.360 [-0.851, 3.43] -0.360 [-0.851, 5.66]
hhfoodsecure
Mean (SD) NA (NA) NA (NA) NA (NA) NA (NA) NA (NA)
Median [Min, Max] NA [NA, NA] NA [NA, NA] NA [NA, NA] NA [NA, NA] NA [NA, NA]
Missing 107 (100%) 88 (100%) 96 (100%) 106 (100%) 397 (100%)
nperson_miss
Mean (SD) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Median [Min, Max] 0 [0, 0] 0 [0, 0] 0 [0, 0] 0 [0, 0] 0 [0, 0]
parity_miss
Mean (SD) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Median [Min, Max] 0 [0, 0] 0 [0, 0] 0 [0, 0] 0 [0, 0] 0 [0, 0]
meducyrs_miss
Mean (SD) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Median [Min, Max] 0 [0, 0] 0 [0, 0] 0 [0, 0] 0 [0, 0] 0 [0, 0]
hhfoodsecure_miss
Mean (SD) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0)
Median [Min, Max] 1.00 [1.00, 1.00] 1.00 [1.00, 1.00] 1.00 [1.00, 1.00] 1.00 [1.00, 1.00] 1.00 [1.00, 1.00]
Control
(N=100)		 BEP+ExBf
(N=100)		 BEP+ExBf+AZT
(N=100)		 Overall
(N=300)
sex
Female 58 (58.0%) 48 (48.0%) 62 (62.0%) 168 (56.0%)
Male 42 (42.0%) 52 (52.0%) 38 (38.0%) 132 (44.0%)
mage
Mean (SD) 24.0 (4.74) 25.2 (5.94) 23.4 (3.64) 24.2 (4.91)
Median [Min, Max] 23.0 [17.0, 35.0] 25.0 [17.0, 37.0] 24.0 [16.0, 35.0] 24.0 [16.0, 37.0]
meducyrs
Mean (SD) 7.46 (2.04) 7.96 (1.92) 8.26 (1.78) 7.89 (1.94)
Median [Min, Max] 8.00 [2.00, 14.0] 8.00 [3.00, 12.0] 8.00 [3.00, 14.0] 8.00 [2.00, 14.0]
nrooms
Mean (SD) 2.74 (1.28) 2.46 (1.51) 2.58 (1.53) 2.59 (1.45)
Median [Min, Max] 3.00 [1.00, 6.00] 2.00 [1.00, 7.00] 2.00 [1.00, 6.00] 2.00 [1.00, 7.00]
parity
Mean (SD) 2.68 (1.77) 3.10 (2.36) 2.42 (1.35) 2.73 (1.89)
Median [Min, Max] 2.00 [1.00, 8.00] 3.00 [1.00, 12.0] 2.00 [1.00, 6.00] 2.00 [1.00, 12.0]
nperson
Mean (SD) 10.2 (3.62) 9.58 (4.42) 10.3 (4.55) 10.0 (4.21)
Median [Min, Max] 10.0 [3.00, 19.0] 9.00 [3.00, 19.0] 10.5 [3.00, 18.0] 10.0 [3.00, 19.0]
imp_water_src
Improved 96 (96.0%) 94 (94.0%) 96 (96.0%) 286 (95.3%)
Unimproved 4 (4.0%) 6 (6.0%) 4 (4.0%) 14 (4.7%)
impfloor
0 2 (2.0%) 2 (2.0%) 4 (4.0%) 8 (2.7%)
1 98 (98.0%) 98 (98.0%) 96 (96.0%) 292 (97.3%)
cookplac
2 (2.0%) 8 (8.0%) 6 (6.0%) 16 (5.3%)
Separate space for cooking 98 (98.0%) 92 (92.0%) 94 (94.0%) 284 (94.7%)
dvseason
Early Summer 8 (8.0%) 6 (6.0%) 14 (14.0%) 28 (9.3%)
Late Summer / Monsoon 30 (30.0%) 28 (28.0%) 32 (32.0%) 90 (30.0%)
Post monsoon / Autumn 44 (44.0%) 38 (38.0%) 26 (26.0%) 108 (36.0%)
Winter 18 (18.0%) 28 (28.0%) 28 (28.0%) 74 (24.7%)
dlvloc
0 26 (26.0%) 20 (20.0%) 32 (32.0%) 78 (26.0%)
1 6 (6.0%) 10 (10.0%) 10 (10.0%) 26 (8.7%)
4 64 (64.0%) 66 (66.0%) 54 (54.0%) 184 (61.3%)
5 4 (4.0%) 4 (4.0%) 4 (4.0%) 12 (4.0%)
hhwealth
Mean (SD) 0.0389 (0.857) -0.0987 (1.04) 0.0598 (1.08) 0.000000000000000137 (0.998)
Median [Min, Max] 0.0158 [-1.68, 2.09] -0.246 [-1.68, 2.13] 0.0942 [-1.68, 1.94] -0.0227 [-1.68, 2.13]
hhfoodsecure
Mean (SD) NA (NA) NA (NA) NA (NA) NA (NA)
Median [Min, Max] NA [NA, NA] NA [NA, NA] NA [NA, NA] NA [NA, NA]
Missing 100 (100%) 100 (100%) 100 (100%) 300 (100%)
nperson_miss
Mean (SD) 0 (0) 0 (0) 0 (0) 0 (0)
Median [Min, Max] 0 [0, 0] 0 [0, 0] 0 [0, 0] 0 [0, 0]
parity_miss
Mean (SD) 0 (0) 0 (0) 0 (0) 0 (0)
Median [Min, Max] 0 [0, 0] 0 [0, 0] 0 [0, 0] 0 [0, 0]
meducyrs_miss
Mean (SD) 0.600 (0.492) 0.540 (0.501) 0.660 (0.476) 0.600 (0.491)
Median [Min, Max] 1.00 [0, 1.00] 1.00 [0, 1.00] 1.00 [0, 1.00] 1.00 [0, 1.00]
hhfoodsecure_miss
Mean (SD) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0)
Median [Min, Max] 1.00 [1.00, 1.00] 1.00 [1.00, 1.00] 1.00 [1.00, 1.00] 1.00 [1.00, 1.00]

(To add unblinded)

Table 2- summary of number and type of HM components

(To add)

(Add internal table on number of missing by component)

Figure 1: heatmaps of primary outcomes

Example from Misame timepoint 2:

Figure 2-4: Arm-specific intervention effects significant after FDR correction

Figure S1-SX: Volcano plots

Figure SX-SX: Volcano plots of intervention effects on HM change

Intervention effects on dimension reduction procedure results

(To add-in progress)

  1. First principal component
  2. SuperLearner weighted outcome score

Variable importance of biomarker category

Importance of removing categories

Importance of adding categories

Intervention effects on growth

(notes: unblinded as sanity check for main)

Negative and positive control results

Use Elicit antibiotics as negative control (they were given to infants after BM collection) and use Elicit Nico as positive control (Vitamin b supplementation that should have an effect specifically on the B-vitamins).

Percent of significant intervention effects across all primary biomarkers: 2.6315789

Postive control plot:

Interactive results explorer

(Publish and link to shiny app)