Table 1.

Studies evaluating the possible effects of maternal nutrition-related factors on HMO content and profiles.

Reference	Exposure Variables of Interest	Study Design	Population and Sample Number	Maternal Nutritional Assessment Tool	Milk Sampling	HMO Analysis	Relevant Main Outcomes
Azad et al. [12]	Maternal pre-pregnancy BMI and usual dietary intake during pregnancy.	Prospective observational study.	n = 427 milk samples, 1 from each mother-infant dyad (healthy term infants, Canadian multi-ethnic mothers).	Validated FFQ during pregnancy; pre-pregnancy BMI (self-reported weight, measured height).	Combined and refrigerated single sample from multiple feeds during a 24-h period at 3–4 months postpartum (median: 16 weeks, IQR: 14–19 weeks).	HPLC; 19 HMOs; total and relative abundance of HMOs; total HMO-bound fucose and sialic acid; FUT2 “Secretor status” defined by 2′-FL.	Maternal dietary intake and BMI not correlated with HMO concentrations.
Davis et al. [14]	Seasonal environmental changes as proxy for caloric intake.	Prospective observational study.	n = 99 milk samples, from 33 mother-infant dyads (rural African Gambian women).	Maternal body weight. Season/environment as proxy for caloric intake.	33 samples × 3 time points (postpartum weeks 4, 16, 20); 5mL of hand-expressed milk from each breast into a separate tube in the morning. Start, middle or end of feed not specified.	Nano-HPLC-Chip/TOF Mass Spectrometry; 19 HMOs. Total HMOs, Fucosylated, sialylated, undecorated HMOs. Mothers with less than Secretors > 6% relative α (1–2) fucosylation.	HMO levels are associated with season. HMO concentrations/total amount significantly higher (p = 0.01) in dry season (food is more plentiful and hence total dietary energy intake may be greater).
Ferreira et al. [15]	Maternal anthropometric characteristics.	Prospective longitudinal observational study.	n = 174 milk samples from 101 subjects (healthy, Latin-American, pregnant women).	Pre-pregnancy BMI (self-reported pre-pregnancy weight and measured height), gestational weight gain (weighed at last prenatal visit); Supplement use via questionnaire (iron and folic acid).	Manual expression of milk samples at 2–8 days, 28–50 and 88–119 days, in the morning after breakfast.	HPLC with fluorescence detection (HPLC-FL); 19 HMOs. HMO absolute (total) and relative abundances. Secretor status determined by presence of 2′-FL and LNFP I.	Maternal pre-pregnancy weight and BMI is associated with HMO composition (low-moderate Spearman correlation values; positively correlated with LNnT (0.4) and inversely correlated with LNFP III (-0.4)).
Gridneva et al. [16]	Maternal body composition indices (fat-free and fat mass, percentage fat mass (%FM).	Longitudinal proof of concept/pilot study.	n = 80 milk samples, from 20 mother-infant dyads (healthy term infants and Australian, predominantly Caucasian mothers).	Maternal body weight, BMI, and body composition indices at 4 visits utilising BIS.	Small (1–2 mL) pre-/post feed milk samples were collected into 5 mL polypropylene vials when infants were 2 and/or 5, 9, and 12 months-old.	None (Total HMO concentration (g/L) estimated by deducting lactose concentration from total carbohydrate concentration). No determination of Secretor status.	No associations with maternal body composition; prior to FDR adjustment, higher HMO calculated daily intake was associated with higher maternal %FM and FM/FFM between 2 and 5 months, and with lower maternal %FM and FM/FFM at 9 and 12 months.
Isganaitiset al. [28]	Maternal obesity and postnatal weight gain.	Prospective observational study.	n = 57 total milk samples; from 31 mothers at 1 month and 26 at 6 months (American, predominantly Caucasian women).	Maternal weight, height, BMI at study site by researchers. Participants grouped by maternal pre-pregnancy BMI (n = 15 BMI < 25, lean, n = 16 BMI ≥ 25 kg/m2, overweight/obese). Gestational weight gain, BMI at 1 month postpartum.	Complete expression of a single breast (right preferred) at 1 and 6 months postpartum using an electric pump. 2–2.5 h after last feed between 8 and 10 am (around midday).	Untargeted metabolomics analysis using LC-GCMS. Reported on 3 oligosaccharides: 2′-FL, LNFP I, and LNFP II/III. No determination of Secretor status.	Maternal obesity was linked to differences in HMO composition at 1 month postpartum (2′-FL, LNFP I and LNFP II/III significantly correlated with maternal BMI).
Jorgensenet al. [18]	Lipid-based Nutrient (LNS) or multiple micronutrient (MMN) supplements, compared with iron and folic acid during pregnancy and placebo postpartum. Potential covariates included baseline maternal BMI (in kg/m2).	Randomised, single (assessor)-blind, parallel group-controlled supplementation trial. Outcomes assessed according to intention to treat principle.	n = 645 breastmilk samples (rural Malawian (African) women, low socio-economic status).	Adherence index for supplement compliance. Weight and height in triplicate by trained anthropometrists at enrollment to calculate BMI during pregnancy (≤20, at 32 and 36 gestational weeks) and once after birth (at 1–2 weeks after delivery).	Single sample, manual expression of the full content of one breast into a sterile plastic cup at 6 months postpartum.	Nano-LC microfluidic chip coupled to electrospray time-of flight mass spectrometer. Summed total HMOs, fucosylated, sialylated and nonfucosylated neutral glycans. No determination of Secretor status.	Supplementation with an LNS or MMN capsule during pregnancy and postpartum did not increase HMO or bioactive milk proteins. No interactions or group differences in HMOs according to maternal BMI.
Larsson, et al. [19]	Maternal pre-pregnancy BMI, gestational weight gain, maternal weight at 5 and 9 months postpartum.	Prospective observational cohort study.	n = 60 milk samples; from 30 mother-infant dyads (13 high-weight infants and 17 normal-weight healthy Danish infants).	Maternal pre-pregnancy BMI and gestational weight gain self-reported; Maternal weight and height measured using standardized procedure at the infant’s age 5- and 9-months visits.	Well-mixed samples of right and left breasts: mothers were asked to pump the entire content of both breasts using a manual breast pump at 5 and 9 months postpartum.	HPLC after fluorescent derivatization; 19 HMOs, total HMO-bound fucose and sialic acid, total HMO. Secretor status was determined based on presence or near-absence of 2′-FL and LNFP I.	Gestational weight gain was not associated with HMO. Maternal BMI at 5 months postpartum was positively with 2′-FL, total HMO and total HMO-bound fucose; and negatively associated with 6′-SL and LSTb (all p ≤ 0.03). Weak associations between HMO and Maternal pre-pregnancy BMI.
McGuire et al. [20]	Maternal anthropometric indices (weight, height, BMI).	Cross-sectional, epidemiologic cohort study that involved multiple (11) international sites.	n = 410 milk samples; 1 from each healthy, breastfeeding woman; multisite: 40, 40, 40, 40, 40, 42, 43, 41, 24, 41, and 19 women from rural Ethiopia, urban Ethiopia, rural Gambia, urban Gambia, Ghana, Kenya, Peru, Spain, Sweden, USA Washington, and USA California.	Maternal body weight, height via questionnaire (self-reported); BMI calculated upon enrolment (2 weeks -5 months postpartum/during lactation).	1 breast only; ≤200 mL (typically 40–60 mL), manually expressed or with a breast pump; at 2 weeks–5 months postpartum.	HPLC-MS; 19 HMOs. Proportion of each HMO and the total concentration of HMOs as the sum of the annotated oligosaccharides. Secretor milk was defined as having a 2′-FL concentration that was greater than a natural, very low break in the data.	Maternal weight and BMI were positively correlated with 2′-FL (r = 0.20), FLNH (r = 0.19 and 0.15, respectively). Maternal weight was positively correlated with LNFP III (r = 0.20) and DFLNT (r = 0.14). Maternal weight and BMI were inversely correlated with LNnT and DSLNT (r = 20.16 and 20.21, respectively; and r = 20.20 and 20.24, respectively).
Moossavi et al. [29]	Maternal body composition (BMI) and fish oil supplement use during pregnancy.	Cross sectional observational study. (Representative cohort from the Longitudinal, population-based birth cohort study (CHILD)).	n = 393, 1 breastmilk sample from representative subset of mothers in the CHILD study (healthy term infants, Canadian multi-ethnic mothers).	BMI calculated (self-reported weight, measured height), Fish oil supplement self-reported by standardized questionnaire.	1 sample at 3–4 months postpartum (mean (SD) 17 (5) weeks postpartum), mix of foremilk and hindmilk from multiple feeds during a 24-h period; manual/hand or pump expression.	HPLC-MS; 19 HMOs, summed to estimate total HMO concentration, HMO-bound fucose (Fuc) and HMO-bound sialic acid (Sia). Maternal secretor status by the presence of 2′-FL or LNFP I.	Maternal diet and BMI are interrelated, and both can modify gut microbiota composition as well as the macro- and micro-nutrient profile and microbiota of human milk (although effect sizes were small (<2% of variation explained).
Qiao et al. [21]	Maternal dietary intake during lactation.	Cross-sectional observational study.	n = 90, 1 breastmilk sample per woman (healthy Chinese women with term infants (37–42 weeks)).	Validated 72-h food frequency questionnaire, weighed where possible; Chinese Dietary Reference Intakes.	1 sample taken at day 40 (±7) postpartum at the end of a breastfeed; 10 mL within 15 mins, stored at −25 °C.	HPLC-FLD; human breast milk sialic acid concentrations (free sialic acid, bound to oligosaccharides and bound to protein). No individual HMO quantification.	82.35% of the sialic acid in breastmilk was found bound to free oligosaccharides. Higher dietary intake of Vitamin A (and of milk, beef, egg, mutton, and pork) was associated with higher milk sialic acid levels (standardized coefficients = 0.713; p = 0.000).
Quin et al. [27]	Maternal dietary intake during lactation.	Prospective cohort clinical study.	n = 16 breastmilk samples, 1 per mother (healthy Euro-Canadian mothers, divided into two groups classified as milk- or almond beverage-consumers).	Self-Administered 24-h (ASA24) diet-recall survey for the 24-h period preceding milk collection.	At 5 months postpartum, manual expression of a few drops of milk (discarded) before collecting 10 mL of foremilk in the morning.	93 (median = 87) HMOs. Quantitation of total reducing sugars, Neu5Ac and Neu5Ac, neutral monosaccharide analysis (fucose, galactose), HMO profiling by CE-LIF, and targeted HMO analysis by HPLC-MS. Determination of secretor status by CE of the median levels of 2′-FL, LDFT, and LNFP I.	In Se+ samples (n = 12), relative levels of Fuc and Gal in HMOs were positively correlated with both the total sugars (p < 0.01) and total dietary fiber (p < 0.05) ingested within the 24-h period prior to milk collection. Several sulfonated/phosphorylated HMOs were positively correlated with breast milk monounsaturated and polyunsaturated fats, and negatively correlated with levels of saturated fats (p < 0.05).
Samuel et al. [8]	Maternal pre-pregnancy body composition (compared the concentrations of HMOs between overweight normal weight women at six different time points over the first four months of lactation, adjusted for milk group).	Longitudinal, observational, multicenter European study (Atlas of Human Milk Nutrients).	n = 1491 milk samples over 6 visits from 290 women (healthy lactating European women from France, Italy, Spain, Romania, Portugal, Sweden, and Norway).	Self-reported maternal pre-pregnancy weight and height to calculate pre-pregnancy BMI (categorized as normal weight: 18.5–24.9 kg/m2 and overweight: 25.0–29.9 kg/m2). Weight loss postpartum (kg).	Samples collected at 3 days, 14 days, 1 month, 2 months, 3 months and 4 months after delivery. Milk collected at 11h00 ± 2h00, electric breast pump, single (same) breast for the entire study (emptied in the previous feed), mixed full breast expression.	Targeted HMO analysis by HPLC-MS, and profiling of 20 HMOs by CE-LIF. Summed total amount of HMOs between milk groups, categorized mothers in one of four groups based on presence of specific α-1,2 and α-1,4- fucosylated HMOs (2′-FL, and LNFP II).	Overweight women (BMI 25.0–29.9 kg/m2) had higher concentrations of 3′SL, 6′GL (p < 0.05). The magnitude of the effect observed were generally low, and for 3′SL and 6′GL 22 and 29 mg/L, respectively.
Seppo et al. [22]	Maternal probiotic supplementation during pregnancy (5 × 109 CFU Lactobacillus rhamnosus GG, 5 × 109 CFU L. rhamnosus LC705, 2 × 108 CFU Bifidobacterium breve Bb99, 2 × 10 9 CFU Propionibacterium freudenreichii ssp. shermanii JS as freeze-dried capsules twice daily).	Randomized, double-blind, placebo-controlled study with 2 parallel groups (probiotic preparation or a placebo for 2 to 4 weeks before delivery, i.e., from 36 weeks’ gestation until the birth).	n = 81 colostrum samples (pregnant women carrying children at increased risk for allergy from the Helsinki suburban area).	Developed questionnaire to assess compliance to the supplementation.	Stored frozen colostrum samples from a previous RCT of probiotic supplementation study of 1223 pregnant women.	HPLC; 19 HMOs. Freezing does not affect HMO levels.	3FL and 3′SL significantly higher in the probiotic group (p = 0.008 and p = 0.006). Levels of DFLNH, LNnT, LNFP, 6′-SL were lower in the supplementation group (p = 0.005, p = 0.01, p = 0.03, p = 0.03). These changes are consistent with a change in select pathways in overall HMO biosynthesis.
Tonon et al. [30]	Maternal anthropometry (pre- and post-pregnancy BMI) and allergic disease status.	Cross-sectional, observational study.	n = 78 mature human milk samples, 1 per mother (Portuguese/Brazilian women, multi-ethnic)	Maternal pre-pregnancy BMI (self-reported weight/obtained from medical records); BMI during lactation (weight and height measured at inclusion, median: 32 days, IQR: 25–46 days postpartum); allergic disease by ISAAC questionnaire.	Manual expression of the breast opposite to the one previously emptied by the infant; 5–15 mL collected in a sterilized glass bottle in the morning (8:30–12:00 am), at 17 to 76 days postpartum (median: 32 days, IQR: 25–46 days)).	LC-MS, performed in duplicate; 16 HMOs (to represent about 90% of the total HMOs in human milk). Secretor and Lewis phenotype of the mothers based on the presence of indicative α1-2 and α1-4 fucosylated HMOs.	Maternal body composition during lactation is associated with concentrations of some HMOs in Se+ women (positive correlation between 2′-FL and maternal BMI (r = 0.30)).

FFQ, food frequency questionnaire; BMI, body mass index; HMOs, Human Milk Oligosaccharides; FUT2, Galactoside 2-alpha-L-fucosyl transferase 2; 2′-FL, 2′-Fucosyllactose; HPLC, high-performance liquid chromatography; LC-GCMS, liquid chromatography–gas chromatography–mass spectrometry; HPLC-FLD, Fluorescence detector-high performance liquid chromatography; MS, mass spectrometry; CE-LIF, Capillary electrophoresis with laser-induced fluorescence detection; Nano-HPLC-Chip/TOF MS, Nano- high-performance liquid chromatography-Chip/Time of Flight mass spectrometry; LNFP, Lacto-N-fucopentaose; LNnT, Lacto-N-Neotetraose; FM, fat mass; FFM, fat-free mass; 6′-SL, 6´Sialyllactose; LSTb, sialyl-lacto-N-tetraose.