Compositional differences between preterm milk of different gestational ages with the term milk: a comparative lipidomic study by LC-MS/MS

doi:10.21203/rs.3.rs-861207/v1

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Compositional differences between preterm milk of different gestational ages with the term milk: a comparative lipidomic study by LC-MS/MS

https://doi.org/10.21203/rs.3.rs-861207/v1

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Background

Studies are beginning to emerge on the important biological effects of the milk lipids exerted on the recipient infant, especially on the premature infants. The aim of this study was to comprehensively describe lipidomic differences between preterm milk of different gestational ages with the term milk over the course of lactation.

Methods

Breast milk samples were collected from 88 mothers giving birth prematurely and 39 mothers delivering at full-term (FT). Lipid profiles were assessed using an LC-MS/MS metabolomics strategy. Orthogonal partial least-squares discriminant analysis (OPLS-DA) and pathway analysis were subsequently performed.

Results

The OPLS-DA score plots significantly distinguished the lipids in preterm milk of different gestation ages from their counterparts in term milk. The concentrations of 10 out of 43 lipid subclasses were found to be persistently higher in preterm compared to term milk over the course of lactation; the diacylglycerol (DAG) and a bioactive subclass fatty acid ester of hydroxyl fatty acid (FAHFA) contributed the most to the differences. In terms of individual lipid species, the ten highest substances found in very preterm (VPT) colostrum compared to FT colostrum mainly come from the phosphatidylethanolamine class and the DAG species. Lipid species from the free fatty acid and FAHFA classes were significantly higher in either extremely preterm (EPT) or VPT mature milk (variable importance in projection > 1, P < 0.0001 for all). The differential lipids between each preterm group and its term counterpart were predicted to be mainly involved in six metabolic pathways, including glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, linoleic acid metabolism, alpha-Linolenic acid metabolism, arachidonic acid metabolism and glycerolipid metabolism.

Conclusions

The lipids in preterm and term milk showed substantial differences, which may be critical for postnatal growth, as well as the neural and immune development of newborns, especially EPT and VPT.

General Biochemistry

Endocrinology & Metabolism

human milk

lipidomic

LC-MS/MS

very preterm

extremely preterm

infants

Premature infants are high-risk groups for growth and neurodevelopmental retardation, as well as infectious diseases, and these are the main causes of death for children under 5 years old. With the continuous progress of perinatal medicine and intensive care technology, increasing numbers of premature infants with short gestational ages and low birth weights are surviving. How to ensure the appropriate growth of premature infants, prevent all kinds of infectious and non-infectious diseases, and improve the prognosis of nerve development while improving survival rate, are all important issues of global concern, and are essential to improving the quality of life and population of children. The World Health Organization (WHO) defines gestational age (GA) ≥ 28 weeks but < 32 weeks as very preterm (VPT), while GA < 28 weeks is defined as extremely preterm (EPT). Although VPT and EPT account for only 2% of all live births and about 16% of all premature infants, the mortality, disease incidence and long-term adverse prognosis of VPT and EPT are much higher than those of moderate preterm (MPT) infants, with a GA ≥ 32 and < 37 weeks [1, 2].

Human milk (HM) is very important for infants, as it provides all the energy and nutrients normal infants require within 6 months. Breastfeeding has multiple benefits for premature infants, such as improved immunity, decreased morbidity and mortality associated with necrotizing enterocolitis, better neurodevelopmental outcomes and improved long-term health outcomes [3–6]. Lipids are one of the most important energy substances in HM, and provide essential vitamins, polyunsaturated fatty acids, complex lipids, and bioactive components to the recipient infants. The biological effects of the lipids provided to breastfeeding infants, for example, on gastrointestinal maturation, lipid and lipoprotein metabolism, membrane composition and function, neurodevelopment and immune function, have also been recognized [7, 8]. Furthermore, the lipid profile of HM impacts early growth in preterm infants [9].

Days postpartum (lactation stage), GA and maternal diet are factors known to influence HM content [10]. Many researchers have already performed dozens of valuable studies on the specific lipid classes in HM, such as triacylglycerol (TAGs) [11, 12] or phospholipids (PLs) [13, 14]. With present-day “omics” techniques, a comprehensive and quantitative analysis of HM lipid composition has become possible. Liquid chromatography tandem–mass spectrometry (LC-MS/MS) is extensively applied in identifying untargeted lipid profiles, due to its high resolution and precision [15]. Both Sokol, E. et al [16] and Hewelt-Belka, W. et al [17] used the comparative LC-MS/MS strategy to investigate the lipidomic differences between HM and formula milk (FM), and found that the lipid composition of FM deviates significantly from that of HM. They also observed significant differences between colostrum and HM collected at other lactation stages.

Metabolomic studies on preterm and full-term (FT) milk have revealed significant differences in the presence of metabolites important for infant development, such as phosphocholine, lysine and glutamine [18]. However, lipidomic studies using LC-MS/MS to compare preterm and FT milk over the course of lactation are still scarce. Ingvordsen Lindahl, I.E. et al. [19] focused on the PL profile of preterm milk (mean GA 30.8 ± 3.4 weeks), and found that both GA and age postpartum affected the 31 PL species found in HM during the first 15 weeks postpartum. Significantly higher concentrations of phosphatidylcholine (PC), sphingomyelin (SM) and total PL were found in preterm milk throughout lactation. Xu, L et al. [20] recently collected six MPT and six FT colostrum samples from healthy breastfeeding mothers, and found that phosphatidylethanolamine (PE) and PC were robustly increased, while diacylglycerol (DAG) and ceramide were markedly decreased, in MPT colostrum. Besides these two studies, data concerning lipid composition differences between preterm and FT milk are remarkably scarce. This knowledge gap must be filled in order to further determine the impacts of these differential lipids on infant growth and development, as well as to provide suitable nutrition for premature infants, particularly EPT and VPT.

In the current study, we conducted an LC-MS/MS-based comparative lipidomic analysis of HM from different GA, using a relatively large sample. Lipid profile differences were assessed by: (i) comparison of VPT and MPT colostrum with its FT counterparts; (ii) comparison of EPT, VPT and MPT mature milk with its FT counterparts. As far as we know, this study is the first to comprehensively describe differences between preterm and term milk lipidomes over the course of lactation.

Ethical Approval

Informed consent was obtained from all subjects involved in the study. Ethical approval for this study was granted by the Ethics Committee of the Children’s Hospital of Fudan University (Children’s Hospital of Fudan University Ethics Protocol 2019–087).

Milk Sample Collection

Preterm milk samples of known GA and lactational stage were collected from non-smoking, non-diabetic mothers in the neonatal intensive care unit of the Children’s Hospital of Shanghai (Shanghai, China). Colostrum (<5 postpartum days, n=20 for VPT, n=20 for MPT) and mature milk samples (4 weeks postpartum, n=11 for EPT, n=23 for VPT and n=21 for MPT) spanning 24/0 weeks to 36/4 weeks of gestation were collected by 88 mothers with preterm (delivered before the 37th week of gestation) infants. The mean age of the mothers was 31.5 years, mean gestational age was 30.25 weeks and mean infant weight at birth was 1470 grams (Table 1). It is difficult to collect sufficient colostrum from EPT mothers, thus the corresponding samples were not collected. FT milk samples were obtained from the Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital (Nanjing, China). Colostrum (n=30) and mature milk samples (n=25) were donated by 39 mothers who had delivered healthy and term (delivered after the 37th week of gestation) infants. Preterm and term samples were kept at −80 °C until analysis.

Metabolites Extraction

As previous studies have described [21, 22], 100 μL of sample was transferred to an EP tube, and mixed with 480 μL of extract solution (MTBE:methanol = 5:1) containing internal standard. After 30 s of vortexing, the samples were sonicated for 10 min in an ice-water bath. Then, the samples were incubated at -40 ℃ for 1 h and centrifuged at 3000 rpm for 15 min at 4 ℃. A total of 350 μL of supernatant was transferred to a fresh tube and dried in a vacuum concentrator at 37 ℃. Then, the dried samples were reconstituted in 100 μL of 50% methanol in dichloromethane by sonication for 10 min in an ice-water bath. The solution was then centrifuged at 13000 rpm for 15 min at 4 ℃, and 90 μL of supernatant was transferred to a fresh glass vial for LC-MS/MS analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all of the samples.

LC-MS/MS Analysis

LC-MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies), equipped with a Kinetex C18 column (2.1 × 100 mm, 1.7 μm, Phenomen). The mobile phase A consisted of 40% water, 60% acetonitrile, and 10 mmol/L ammonium formate. The mobile phase B consisted of 10% acetonitrile and 90% isopropanol, which was mixed with 50 mL of ammonium formate, added at 10 mmol/L for every 1000 mL of mixed solvent. The analysis was performed with the following elution gradient: 0~12.0 min, 40~100% B; 12.0~13.5 min, 100% B; 13.5~13.7 min, 100~40% B; 13.7~18.0 min, 40% B. The column temperature was 55 ℃. The auto-sampler temperature was 4 ℃, and the injection volume was 2 μL (pos) or 4 μL (neg) [21]. The QE mass spectrometer was used as it can acquire MS/MS spectra in the data-dependent acquisition (DDA) mode under the control of the acquisition software (Xcalibur 4.0.27, Thermo). In this mode, the acquisition software continuously evaluates the full-scan MS spectrum. The ESI source conditions were as follows: sheath gas flow rate 30 Arb, Aux gas flow rate 10 Arb, capillary temperature 320 ℃ (positive) and 300 ℃ (negative), full MS resolution 70000, MS/MS resolution 17500, collision energy 15/30/45 in NCE mode, spray voltage 5 kV (positive) or –4.5 kV (negative) [23, 24].

Data preprocessing and annotation

The raw data files were converted to the mzXML format using the “msconvert” program from ProteoWizard. Peak detection was first applied to the MS1 data. The CentWave algorithm in XCMS was used for peak detection in the MS/MS spectrum, while lipid identification was achieved through spectral matching using the LipidBlast library. We applied orthogonal partial least-squares discriminant analysis (OPLS-DA) to the unit variance-scaled spectral data so as to visualize the differences between preterm and term milk. The coefficient loading plots and variable importance in projection (VIP) of the OPLS-DA model were employed for identifying the spectral variables related to sample dissolution. The VIP scores ranked the components according to their role in the observed separation [24, 25]. Statistical distinctness was established using Student’s t-test, with P < 0.05 signifying significance. We conducted pathway assessments in MetaboAnalyst (http://www.metaboanalyst.ca/).

Multivariate statistical analysis of lipids

Based on the OPLS-DA, the score scatter plot indicates a distinct separation between each preterm group and its term counterpart. The sample is basically within the 95% confidence interval (Hotelling’s T-squared ellipse) (Figures 1-5). The R²Y and Q²Y values in the OPLS-DA model were all > 0.5 (in the comparison of VPT colostrum and FT colostrum, R²Y = 0.829 and Q² = 0.665 in the negative ionization mode, and R²Y = 0.817 and Q² = 0.644 in the positive ionization mode; in the comparison of MPT colostrum and FT colostrum, R²Y = 0.88 and Q² = 0.788 in the negative ionization mode, and R²Y = 0.851 and Q² = 0.756 in the positive ionization mode; in the comparison of EPT mature milk and FT mature milk, R²Y =0.822 and Q² = 0.747 in the negative ionization mode, and R²Y = 0.776 and Q² = 0.733 in the positive ionization mode; in the comparison of VPT mature milk and FT mature milk, R²Y =0.771 and Q² = 0.684 in the negative ionization mode, and R²Y = 0.718 and Q² = 0.597 in the positive ionization mode; in the comparison of MPT mature milk and FT mature milk, R²Y = 0.804 and Q² = 0.713 in the negative ionization mode, and R²Y = 0.788 and Q² = 0.758 in the positive ionization mode), implying that the OPLS-DA model was well-determined. These OPLS-DA models were further validated using permutation tests of training sets of 100 times. The R²Y and Q² values were the greatest in the current sample clustering approach, and the longitudinal intercept of the Q² regression line is less than zero. Therefore, the original model has good robustness, without overfitting (Figures 1-5).

Lipid profiling differences between preterm and term HM

Based on the results of the LC-MS/MS analysis, more than nine thousand lipid substances were found in the HM sample. In general, 113-272 lipids (VIP > 1, p < 0.05) were remarkably different when comparing each preterm group with its term counterpart. All of the markedly different lipids are shown in Additional file 1: Table S1-S2. For each group, we calculated the Euclidean distance matrix for the quantitative values of differential lipids, and clustered the differential lipids using the complete linkage method, which we then displayed as a heatmap (see Additional file 2: Figures S1-S10). The top 10 most noticeably up-modulated and down-modulated lipids in each preterm arm in positive ion mode and negative ion mode respectively were listed in Additional file 3: Table S3-S6.

As previously mentioned, we were particularly interested in the unique characteristics of EPT and VPT milk in terms of lipid composition. After eliminating the lipids identified in both the positive, as well as the negative ion mode repeatedly, we found that the top 10 substances that were higher in the VPT colostrum than in the FT colostrum were mostly from the PE class (18:1/22:1, 18:1/20:1, 14:1/26:4 and 16:0/16:0) and the DAG species (12:0/22:5, 16:0/22:6 and 14:1/18:1) (Table 2). In terms of mature milk, lipids from free fatty acid (FA) (18:1, 16:2, 17:3 and 19:2) and a bioactive class fatty acid ester of hydroxyl fatty acid (FAHFA) (16:1/22:4, 22:3/22:4, 2:0/17:2 and 20:2/20:1) constituted the 10 most upregulated substances in the EPT group, as compared with their FT counterparts (Table 3); similarly, lipids from FA (16:2, 17:3 and 18:1) and FAHFA (18:2/14:1, 16:1/22:4, 20:3/22:5, 12:0/18:2 and 18:1/18:2) constituted the 10 most upregulated substance in the VPT group, compared with their FT counterparts (Table 4).

The differences in the comparative proportion of identified lipid species

The differences in the proportions of identified lipid species were used to reveal their composition and distribution. After combining the positive and negative ionization modes, among the 43 subclasses of lipids, we found that the concentrations of acylcarnitine (ACar), ceramide alpha-hydroxy fatty acid-phytospingosine (Cer/AP), ceramide alpha-hydroxy fatty acid-sphingosine (Cer/AS), DAG, Diacylglyceryl trimethylhomoserine (DGTS), FA, FAHFA, lysophosphatidylethanolamine (LPE), monoacylglycerol (MAG), PC and PE were significantly higher in both the VPT and MPT colostrum than in the FT counterparts (Figures 6). The differences in mature milk are generally consistent with those in colostrum, but the level of DGTS did not increase significantly in each premature group compared with the FT counterparts (Figures 7). The two most significantly upregulated lipid clusters in the preterm arm were FAHFA and DAG, in both colostrum and mature milk (Figures 6 and 7).

The Metabolomic Pathways of the Differential Lipids Between the Term and Preterm Milk

Pathway analysis was performed using MetaboAnalyst to explore the potential functions of the significantly different lipids. These lipids were predicted to be involved in six metabolic pathways, including glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, linoleic acid metabolism, alpha-Linolenic acid metabolism, arachidonic acid metabolism and glycerolipid metabolism (Figure 8).

Advances in metabolomics and lipidomics using LC-MS/MS have enabled the accurate quantification of complex lipid molecules in milk, as well as in very small amount of infant serum or plasma [26-29]. Such lipidomic measurements can provide markers for tissue composition [30], and have been proven to be related to many important clinical endpoint events in children and adults [31-33]. Therefore, using these sophisticated and detailed analytical methods in conjunction with appropriate bioinformatics strategies should provide a better understanding of the physiological role of complex lipids in early life.

According to our LC-MS/MS results, in terms of the total contents of major lipid subsets, the concentrations of 10 out of the 43 lipid subclasses were found to be persistently higher in preterm milk compared to term milk over the course of lactation, with the FAHFA and DAG classes contributing the greatest differences. The difference between preterm milk of different GA stages and term milk is relatively consistent; in other words, the composition of preterm milk did not visibly resemble that of term milk as GA increased. In terms of individual lipid species, differential lipids (VIP > 1, p < 0.05) between preterm and term milk began to show discrepancies with increasing GA of the neonate and the progression of lactation. As mentioned in the background section, we paid special attention to EPT and VPT milk. Interestingly, compared with their FT counterparts, several PLs from the PE class (18:1/22:1, 18:1/20:1, 14:1/26:4 and 16:0/16:0) and the DAG species (12:0/22:5, 16:0/22:6 and 14:1/18:1) in VPT colostrum were significantly higher. In mature milk, FA and FAHFA lipids comprised the 10 most upregulated substances in both the EPT and VPT groups (P<0.0001 for all).

DAG is one of the primary lipid subpopulations in living systems, and acts as a second messenger in multicellular activities. It has been reported that DAG can accelerate the β-oxidation of fatty acids and affect the expression of genes related to lipid metabolism, thereby reducing TAG levels in serum and liver [34, 35]. In our current study, we found the total concentrations of DAG to be markedly increased in both preterm colostrum and mature milk, and several individual DAG species, such as DAG 12:0/22:5, 16:0/22:6 and 14:1/18:1 were significantly elevated in the VPT colostrum. The high levels of DAG species in preterm human milk may be linked to enhanced long-term health outcomes; for instance, reduced obesity, diabetes, and cardiovascular disease. Interestingly, Xu, L et al. [20] reported that DAG levels were significantly reduced in the colostrum of mothers who gave birth prematurely. Discrepancies between the present study and that of Xu, L et al. might stem from the significantly smaller sample size (n= 6, in both the preterm group and term groups) and greater GA of premature delivery (32–36 weeks) in the latter. More research is needed to validate our findings.

FAHFAs have come to be recognized as one of the bioactive lipids present in the HM with anti-inflammatory, anti-diabetic effects; they enhance glucose tolerance and the secretion of insulin and glucagon-like peptide 1 (GLP-1) while reducing inflammation [36-39]. Lee, J. et al. [40] also reported the role of FAHFAs in the gut, regulating both innate and adaptive immune responses in a model of colitis. So far, FAHFAs have only been detected in the serum and adipose tissue of mice and humans [36], and adipose tissue represents a major site for FAHFAs synthesis [36, 37]. Brezinova M et al. [41] performed a lipidomic analysis of milk samples obtained from lean and obese mothers, and compared the PAHSA (an FAHFA species containing palmitic acid) levels of these groups. They found, for the first time, that lipids from the FAHFA family exist in human colostrum/transient milk, and that the concentration of FAHFA in HM was negatively correlated with maternal weight. As can be seen from the above, FAHFAs hold great biological significance, but little research has been done on FAHFAs in HM, except the above. For the first time, we found that the levels of FAHFAs in preterm HM at different GAs were significantly higher than in term HM, with the levels of several FAHFA species (16:1/22:4, 22:3/22:4, 2:0/17:2, 20:2/20:1, 18:2/14:1, 16:1/22:4, 20:3/22:5, 12:0/18:2 and 18:1/18:2) remarkably higher in either EPT or VPT mature milk compared with their FT counterparts. Our findings may indicate that FAHFAs play an important role in improving the metabolism and promoting the intestinal maturation of premature infants. More maternal–child cohort studies and animal experiments will be needed to further explore the role and the mechanism of action of FAHFAs in HM.

PLs are a class of lipids with important biological functions, despite only accounting for 1% of total milk lipids [42]. They are involved in the digestion, absorption and transport of TAG, making up the milk fat globule membrane and providing important bioactive components, such as choline, and the long-chain polyunsaturated fatty acids fatty acids (LC-PUFAs) docosahexaenoic acid (DHA) and arachidonic acid (AA), which are crucial for the neural and visual development and growth of the neonate [43-46]. The glycerophospholipids (GPL) PC and PE, and the sphingolipid (SL) SM, are the three major PL classes found in HM. So far, omics has rarely been used to thoroughly study the PLs in both preterm and term milk from different gestation ages and lactation stages.

Here, we show that the total concentration of PE was increased in both preterm colostrum and mature milk, and several individual PE species, such as PE 18:1/22:1, 18:1/20:1, 14:1/26:4 and 16:0/16:0, were significantly elevated in the VPT colostrum. PE, also known as cephalin, is an important part of the nerve cell membrane. It regulates all the metabolic activities of nerve cells and affects a series of important functions of nervous tissue, such as cell permeability, myelin formation and mitochondrial operation [7]. Our results highlight the important role that the PE in HM plays in the early nervous system development of premature infants. Furthermore, LC-PUFAs are often found as FA moieties of PE, and contribute to the neurological as well as visual development of the neonate [47,48]. The transport of LC-PUFA from mother to infant mainly occurs through the placenta in late pregnancy. The dramatically higher content of PE observed in both preterm colostrum and mature milk in our findings suggests that the PE in HM may be an important channel through which premature infants with low GAs can obtain sufficient LC-PUFA through breastfeeding. Furthermore, in the present study, the observed differences in PE between preterm and term colostrum were almost equally prominent in mature milk, which diverges from the observations of Ingvordsen Lindahl, I.E. [19], wherein the effects of gestation subsided in mature milk. Discrepancy might be influenced by the sample preparation process, especially the addition of different internal standards prior to lipid extraction. More homogenization and standardized HM lipidomic studies starting from sample collection and storage are needed in the future.

In addition, we found that preterm colostrum and mature milk contain higher concentrations of PC than their term counterpart. PC is an important source of choline, along with SM, accounting for approximately 17% of the choline in HM [49]. Research has shown that choline is essential to fetal development [50,51]. The three- to fourfold higher choline concentration in fetal vs. term infant plasma corresponds to the fetus’s higher growth rate and choline requirements [52,53]. Plasma choline, however, rapidly and prematurely decreases after preterm birth [52]. Low plasma choline levels may contribute to the impaired lean body mass growth of preterm infants. Recently, in a randomized controlled trial conducted by Bernhard W et al. [18], supplementation with 30 mg/kg/day additional choline increased plasma choline to near-fetal concentrations and improved DHA homeostasis in preterm infants. High concentrations of choline in human breast milk ensure the adequate supply of choline to the breastfed preterm infant, which help to preserve fetal concentrations of plasma choline in the perinatal period [18], thus playing a key role in tissue muscle accumulation, as required for the catch-up growth of premature infants after birth.

Preterm delivery interrupts the normal placental transfer and fetal accretion of choline, complex fatty acids, and phospholipids and fats that occur in the third trimester. Our findings offer useful information for the utilization of these critical constituents as nutritional, as well as functional, factors in infant formula, especially for EPT and VPT. Our metabolomic pathway analysis results provide novel insights into the mechanisms of human milk lipids’ actions in neonatal development.

The vacancy of EPT colostrum samples is a substantial limitation to this study. Further explorations of HM composition in preterm infants will help in the more effective application of nutritional support strategies for preterm infants, especially EPT and VPT.

GA: gestational age; VPT: very preterm; EPT: extremely preterm; MPT: moderate preterm; HM: human milk; TAG: triacylglycerol; PL: phospholipids; LC-MS/MS: liquid chromatography tandem–mass spectrometry; FM: formula milk; FT: full-term; PC: phosphatidylcholine; SM: sphingomyelin; PE: phosphatidylethanolamine; DAG: diacylglycerol; OPLS-DA: orthogonal partial least-squares discriminant analysis; VIP : variable importance in projection; FA: free fatty acid; FAHFA: fatty acid ester of hydroxyl fatty acid ; Acar: acylcarnitine; Cer/AP: ceramide alpha-hydroxy fatty acid-phytospingosine; Cer/AS: ceramide alpha-hydroxy fatty acid-sphingosine; DGTS: Diacylglyceryl trimethylhomoserine; LPE: lysophosphatidylethanolamine; MAG: monoacylglycerol ; GPI: glycosylphosphatidylinositol; MFGM: milk fat globule membrane; GLP-1: glucagon-like peptide 1; LC-PUFA: long-chain polyunsaturated fatty acids fatty acids; DHA: docosahexaenoic acid ; AA: arachidonic acid; GPL: glycerophospholipids; SL: sphingolipid.

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Ethics Committee of the Children’s Hospital of Fudan University (Children’s Hospital of Fudan University Ethics Protocol 2019–087). Informed consent was obtained from all subjects involved in the study.

Consent for publication

Not applicable

Availability of data and materials

The dataset(s) supporting the conclusions of this article is(are) included within the article (and its additional file(s)).

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by the Young Clinical Scientist Program of Fudan Academy of Pediatrics (grant no. EK112520180307).

Authors' contributions

PD and YCZ conceived and designed the study. CXL, YCZ, DYY collected the breast milk samples. PD and YCZ supervised the laboratory detection and bioinformatic analysis. YZ collected and analyzed the clinical data. PD interpreted the results and wrote the original draft. YCZ, CXL and YZ reviewed and edited the draft. PD acquired the funding. All authors read and approved the final manuscript.

Acknowledgments

The authors would like to acknowledge Dr. Chen-bo Ji from the Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital (Nanjing, China) who kindly helped us organize the collection of full-term breast milk. We thank all of the mothers and infants who kindly donated their milk. We thank the assistance from Shanghai Biotree Biotech Co., Ltd.

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Table 1

Baseline characteristics of the infants and breastfeeding mothers
	Colostrum (< 5 postpartum days)						Mature milk (4 weeks postpartum)
	VPT (n = 20)	MPT (n = 20)	FT (n = 30)	P-value			EPT (n = 11)	VPT (n = 23)	MPT (n = 21)	FT (n = 25)	P-value
Gestational age(mean (SD)) Range (weeks)	30.13(1.08) 28.43–31.86 1511(273) 1050–1920 13(65%) 31(4) 25–37 22.22(4.11) 17.72–31.13 95.60%	34.14(1.25) 32.29–36.43	39.10(1.11) 37.71–41.29		< 0.001 < 0.001 0.544 0.631 0.738 < 0.001	26.49(1.16) 24.00-27.86		29.76(0.89) 28.14–31.71	32.78(0.67) 32.00-34.14	38.77(0.92) 37.00-41.57	< 0.001 < 0.001 < 0.05 0.891 0.522 < 0.001
Birth weight(mean (SD)) Range (g)		1916(322) 1150–2445	3370(339) 2600–4251			973(210) 800–1420		1380(170) 1080–1730	1545(335) 910–2300	3322(314) 2557–4110
Male(n (%))		12(60%)	17(57%)			6(55%)		15(65%)	8(38%)	13(52%)
Maternal age(mean (SD)) Range (years)		29(4) 23–38	29(4) 23–38			33(5) 28–45		32(4) 25–40	33(4) 24–40	31(5) 21–42
Maternal BMI at birth(mean (SD)) Range (kg/m²)		22.01(4.19) 17.15–29.98	21.9(3.74) 18.00-28.03			22.67(4.45) 18.15–28.03		21.55(3.28) 18.01–31.13	22.97(4.09) 17.78–30.06	22.11(4.44) 17.50-31.11
Cesarean section (%)		87.33%	33.44%			99.91%		97.44%	87.47%	36.35%
EPT: extremely preterm; VPT: very preterm; MPT: moderately preterm; FT: full-term.

Table 2

The top 10 most notably upregulated lipids in VPT colostrum as compared with FT counterpart
Name	VPT	FT	VIP	P-value
Upregulated
PE(18:1/22:1)	0.02666	0.01349	1.58808	1.37E-07
PE(18:1/20:1)	0.14709	0.0775	1.53485	1.19E-06
DAG(12:0/22:5)	0.02751	0.01009	1.33663	1.19E-06
ACar(18:3)	0.00611	0.00229	1.09655	1.24E-06
DAG(16:0/22:6)	0.06557	0.02807	1.21894	2.47E-06
PE(14:1/26:4)	0.06333	0.0384	1.15569	3.11E-06
PE(16:0/16:0)	0.0386	0.02178	1.05767	6.90E-06
DAG(14:1/18:1)	0.4422	0.08157	1.29495	8.06E-06
FAHFA(16:2/26:4)	2.18617	0.39658	1.5826	8.59E-06
HexCer/NS(d14:3/19:1)	0.01103	0.00453	1.57603	9.98E-06
VPT: very preterm; FT: full-term; VIP: variable importance in projection of the OPLS-DA model

Table 3

The top 10 most notably upregulated lipids in EPT mature milk as compared with FT counterpart
Name	EPT	FT	VIP	P-value
Upregulated
FA(18:1)	0.70866	0.51798	1.64705	5.23E-08
FA(16:2)	0.70769	0.17423	1.87897	6.32E-08
GlcADG(12:0/17:2)	0.02729	0.00664	1.74735	2.48E-07
FAHFA(16:1/22:4)	0.007	0.00207	1.67575	1.51E-06
FA(17:3)	0.02023	0.00502	1.82093	2.24E-06
DAG(18:0/20:4)	0.0167	0.00257	2.48566	3.90E-06
FAHFA(22:3/22:4)	0.01687	0.00354	1.69943	6.28E-06
FA(19:2)	0.10731	0.03116	1.76184	2.96E-05
FAHFA(2:0/17:2)	0.12484	0.04769	1.68875	0.000101
FAHFA(20:2/20:1)	0.01762	0.00357	1.65397	0.000124
EPT: extremely preterm; FT: full-term; VIP: variable importance in projection of the OPLS-DA model

Table 4

The top 10 most notably upregulated lipids in VPT mature milk as compared with FT counterpart
Name	VPT	FT	VIP	P-value
Upregulated
GlcADG(12:0/17:2)	0.02755	0.00664	1.90786	7.50E-09
FA(16:2)	0.60226	0.17423	1.93359	4.25E-08
FAHFA(18:2/14:1)	0.00846	0.00097	1.89332	4.59E-07
Cer/AS(d19:3/16:2)	0.10244	0.0128	1.81358	7.13E-07
FAHFA(16:1/22:4)	0.00616	0.00207	1.79913	8.28E-07
FAHFA(20:3/22:5)	0.05209	0.01026	1.93091	1.35E-06
FA(17:3)	0.01654	0.00502	1.91319	1.4E-06
FA(18:1)	0.66552	0.51798	1.44257	1.77E-06
FAHFA(12:0/18:2)	0.05398	0.00845	1.82852	1.91E-06
FAHFA(18:1/18:2)	0.71503	0.13136	1.80522	1.97E-06
VPT: very preterm; FT: full-term; VIP: variable importance in projection of the OPLS-DA model

FigureS1heatmapVPTFTcolostrumneg.pdf
Figure S1. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in VPT vs. FT colostrum in the negative ionization mode
FigureS2heatmapVPTFTcolostrumpos.pdf
Figure S2. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in VPT vs. FT colostrum in the positive ionization mode
FigureS3heatmapMPTFTcolostrumneg.pdf
Figure S3. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in MPT vs. FT colostrum in the negative ionization mode
FigureS4heatmapMPTFTcolostrumpos.pdf
Figure S4. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in MPT vs. FT colostrum in the positive ionization mode
FigureS5heatmapEPTFTmaturemilkneg.pdf
Figure S4. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in MPT vs. FT colostrum in the positive ionization mode
FigureS6heatmapEPTFTmaturemilkpos.pdf
Figure S6. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in EPT vs. FT mature milk in the positive ionization mode
FigureS7heatmapVPTFTmaturemilkneg.pdf
Figure S7. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in VPT vs. FT mature milk in the negative ionization mode
FigureS8heatmapVPTFTmaturemilkpos.pdf
Figure S8. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in VPT vs. FT mature milk in the positive ionization mode
FigureS9heatmapMPTFTmaturemilkneg.pdf
Figure S9. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in MPT vs. FT mature milk in the negative ionization mode
FigureS10heatmapMPTFTmaturemilkpos.pdf
Figure S10. The heatmap of hierarchical clustering analysis of significantly upregulated and downregulated lipids in MPT vs. FT mature milk in the positive ionization mode
TableS1NEGDifferentiallyExpressedMetabolites.xlsx
Table S1. All of the markedly different lipids when comparing each preterm group with the term counterpart in the negative ionization mode
TableS2POSDifferentiallyExpressedMetabolites.xlsx
Table S2. All of the markedly different lipids when comparing each preterm group with the term counterpart in the positive ionization mode
TableS3Top10lipidsincolostrumneg.docx
Table S3. Top 10 most notably upregulated and downregulated lipids in colostrum of different preterm and term groups in the negative ionization mode
TableS4Top10lipidsincolostrumpos.docx
Table S3. Top 10 most notably upregulated and downregulated lipids in colostrum of different preterm and term groups in the negative ionization mode
TableS5Top10lipidsinmaturemilkneg.docx
Table S5. Top 10 most notably upregulated and downregulated lipids in mature milk of different preterm and term groups in the negative ionization mode
TableS6Top10lipidsinmaturemilkpos.docx
Table S6. Top 10 most notably upregulated and downregulated lipids in mature milk of different preterm and term groups in the positive ionization mode

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Compositional differences between preterm milk of different gestational ages with the term milk: a comparative lipidomic study by LC-MS/MS

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