2-Fucosyllactose in human breast milk can predict the severity of necrotizing enterocolitis

2-Fucosyllactose in human breast milk can predict the severity of necrotizing enterocolitis | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 17 January 2025 V1 Latest version Share on 2-Fucosyllactose in human breast milk can predict the severity of necrotizing enterocolitis Authors : Santosh Navi , Savita Attri 0000-0002-1783-9094 [email protected] , Sourabh Dutta , Ajay Patial , and Jayakanthan Doss Authors Info & Affiliations https://doi.org/10.22541/au.173712979.90594950/v1 321 views 152 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract SCOPE Necrotizing enterocolitis is a serious and life-threatening condition affecting very low birth weight neonates. The composition of human milk oligosaccharides in breast milk varies significantly. This nested case-control study aimed to evaluate the association between levels of the HMO 2-fucosyllactose in breast milk and the occurrence and severity of NEC. METHODS AND RESULTS A total of 247 mother-infant dyads, between 26 and 32 weeks of gestation, were enrolled over a one-year period in a nested case-control study. Breast milk samples were collected between the 9th and 15th day of life. Clinical data for enrolled infants were reviewed at discharge or at six weeks of life. Each case was matched with five controls (1:5). Only matched samples were analyzed for 2’-fucosyllactose (2-FL) levels using LC-MS/MS. The median 2-FL levels in cases were significantly lower compared to controls (p < 0.03). However, univariate analysis did not show a significant association. An inverse correlation was observed between NEC severity and 2’-FL levels. Notably, 2’-FL levels below 311 µg/mL demonstrated a sensitivity of 80% in predicting NEC occurrence. 2. CONCLUSION Studies are needed to explore the role of 2’-FL in NEC prevention and its potential as a supplement. Title Page Title of the article: 2-Fucosyllactose in human breast milk can predict the severity of necrotising enterocolitis 2. The names of all authors: (first name, middle initial, last name) Santosh Navi, Savita Verma Attri, Sourabh Datta, Ajay Patial, Jayakanthan Kabeerdoss Department of Pediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh, India-160012. Corresponding Author: Prof. Savita Verma Attri, Department of Pediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh, India-160012. Email: [email protected] , M: +91-9872642288 Abbreviations: 2’-Fucosyl lactose (2-FL), Human milk oligosaccharides (HMOs), Necrotizing enterocolitis (NEC), Neonatal intensive care unit (NICU). Key words: Human breast milk, necrotizing enterocolitis, neonatal intensive care, HMOs. Abstract SCOPE Necrotizing enterocolitis is a serious and life-threatening condition affecting very low birth weight neonates. The composition of human milk oligosaccharides in breast milk varies significantly. This nested case-control study aimed to evaluate the association between levels of the HMO 2-fucosyllactose in breast milk and the occurrence and severity of NEC. METHODS AND RESULTS A total of 247 mother-infant dyads, between 26 and 32 weeks of gestation, were enrolled over a one-year period in a nested case-control study. Breast milk samples were collected between the 9th and 15th day of life. Clinical data for enrolled infants were reviewed at discharge or at six weeks of life. Each case was matched with five controls (1:5). Only matched samples were analyzed for 2’-fucosyllactose (2-FL) levels using LC-MS/MS. The median 2-FL levels in cases were significantly lower compared to controls (p < 0.03). However, univariate analysis did not show a significant association. An inverse correlation was observed between NEC severity and 2’-FL levels. Notably, 2’-FL levels below 311 µg/mL demonstrated a sensitivity of 80% in predicting NEC occurrence. CONCLUSION Studies are needed to explore the role of 2’-FL in NEC prevention and its potential as a supplement. MAIN BODY Introduction Necrotizing enterocolitis (NEC) is one of the most life-threatening gastrointestinal emergencies affecting very low birth weight neonates in the neonatal intensive care unit (NICU), with a reported mortality rate of around 30% (1,2). The global incidence of NEC among very low birth weight babies is estimated to be approximately 7% (3). The etiopathogenesis of NEC is complex and multifactorial, with intestinal inflammation, genetic susceptibility, and alterations in the gut microbiome being the most important contributing factors (4, 5). Various studies have shown that formula-fed infants are at a 6- to 10-fold higher risk of developing NEC compared to human milk-fed infants (6). Human milk contains various bioactive components that protect the infant from pathogenic organisms, promote intestinal immune development, and support healthy gut microbiota (7). A major bioactive component of human milk is human milk oligosaccharides (HMOs). HMOs are complex glycans that represent the third most abundant component of human milk (8). HMOs prevent the colonization of pathogenic organisms in the gut, serving as prebiotic substrates, particularly for specific commensal bacteria such as Bifidobacterium. Moreover, HMOs upregulate the gut innate immune system of the infant (9,10). The HMO content has been estimated to range between 1–10 g/L in mature milk and 15–23 g/L in colostrum. Approximately 35–50% of HMOs in breast milk are fucosylated, 42–55% are non-fucosylated neutral, and the remainder are sialylated HMOs (11). Among the various HMOs in breast milk, the most abundant oligosaccharide is 2-fucosyllactose (2-FL), with a median concentration of 2.8 g/L in the first 30 days of life (12). 2-FL protects neonates from NEC by decreasing inflammation through the downregulation of toll-like receptors and enhancing mesenteric perfusion, as demonstrated in animal experiments (13). However, it has been observed that some infants develop NEC despite being predominantly fed human milk, emphasizing the diversity in the composition of milk oligosaccharides (14, 15). This led us to hypothesize that human milk-fed infants who develop NEC have lower levels of 2-FL compared to human milk-fed infants who do not develop NEC. There are limited options available for pediatricians to predict NEC in preterm babies. In this context, the promising role of 2-FL in preventing NEC in animal models is noteworthy (16). However, there is a paucity of literature exploring the role of 2-FL in humans in preventing NEC. This led us to undertake this study to: a) Determine the level of 2-FL in the breast milk of mothers with preterm babies who develop NEC and compare it with the levels in breast milk of mothers with preterm babies who do not develop NEC. b) Explore the association between the levels of 2-FL in human breast milk and the severity of NEC. c) Assess the predictive value of 2-FL levels in human breast milk in mothers of preterm babies (28 to 32 weeks) for the development of NEC. EXPERIMENT SECTION Patient enrolment and sample collection This study was cleared by the Institute Ethics Committee vide reference number NK/7211/MD/544 and a written informed consent was obtained from the mothers of all the babies enrolled. A nested case-control study was designed, enrolling all preterm babies born between 26 and 32 weeks of gestation in the neonatal nursery and NICU, along with their mothers, between July 2021 and July 2022. A total of 3–5 mL of breast milk was collected from each mother enrolled in the study on the 9th–15th day of postnatal life using clean containers. The samples were stored at -80°C until processing. Among all the enrolled subjects, those who developed NEC were labelled as cases, while those who did not develop NEC were considered controls. The babies were reviewed at the time of discharge or at six weeks of life, with all records assessed for the presence or absence of NEC. In cases where NEC was identified, its severity was assessed using a modified Bell’s scoring system. Each case was matched with five controls based on gestational age and birth weight (±100 g). The breast milk samples of only the matched cases and controls were analysed for 2-FL levels. Measurement of 2-FL levels in breast milk Preparation of stock solution The stock solution of 2-FL was prepared in water at a concentration of 1mg/ml and stored at -80°C until used. Working calibration standards were prepared in the range of 78 µg/ml to 10000 µg/ml in water freshly with every batch. Sample preparation 10 µl of milk sample was mixed with 90 µl ultrapure water and was later centrifuged at 14000 rpm for 10 min at room temperature to remove fat from the sample. The aqueous layer was transferred to a fresh micro centrifuge tube and was mixed with 180 µl of ethanol to precipitate the proteins at -80⁰C for 1.5 hours followed by centrifugation at 14000 rpm at room temperature for 15 min. The upper layer was transferred to a fresh microcentrifuge tube and further dried in a vacuum concentrator at 40⁰C for 40 min. The dried residue was suspended in 150µl of ultrapure water and 10µl was injected into LC-MS/MS for analysis. The chromatographic separation and mass spectrometric analysis were performed using Nexera X2 LC (Shimadzu) and QTRAP 4500 mass spectrometer (Sciex) coupled with electrospray ionization (ESI). The chromatographic separation was achieved using Waters’ XBridge BEH amide column (100 x 4.6 mm x2.5µm) kept in a thermostatic column compartment at 45°C. The mobile phase consisted of two components, 5mM ammonium acetate (aqueous) and 80% ethanol v/v with 5mM ammonium acetate (aqueous). Gradient elution was achieved at a flow rate of 0.5 mL/min. The autosampler temperature was kept at 15°C throughout the experiment. The following mass spectrometer parameters were used: source temperature (550°C), ion-spray voltage (4000 V), curtain gas (25 PSIG), ion source gas 1 (50 PSIG), and 2 (50 PSIG). The ions were detected in negative ionization mode by multiple reaction monitoring (MRM) using unit resolution at both quadrupoles. Analyst software version 1.6.2 was used for quantitation. The calibration range for 2-FL was 78-10,000 µg/ml. The limit of detection (LOD) [defined as S/N ratio (Signal-to-Noise ratio>5)] was found to be 0.78 µg/ml. The R 2 coefficient of determination in linear regression was 0.9995. Statistical analysis: MS Excel and SPSS were used for analysing the data. Categorical data were described as frequency percentile. The normality of the distribution of numerical variables was determined using the skewness statistics and Shapiro-Wilk plot. Normally distributed variables were described as mean (SD) and variables with skewed distribution as median (1 st , 3 rd quartile). Subjects were grouped into having NEC and no NEC, and were matched for gestational age with a margin of one week (+/-) and birth weight with a margin of +/-100g. The normally distributed variables were compared between the groups by student t-test and those with skewed distribution by Mann Whitney U test. Categorical variables were compared using the chi-square test with continuity correction or Fischer exact test as applicable. Uni-variate analysis of all predictor variables was performed, and the results were expressed as p values. We performed multi variable logistic regression analysis with NEC as the binary outcome variable 2-FL as the predictive variable and other prognostic factors of NEC as co-variables, those were significant in uni-variate analysis. We generated ROC curves with 2-FL as the index test and NEC as the outcome variable and we determined the area under ROC with 95% CI. RESULTS Out of all enrolled mother-neonate Dyads, median gestational age of neonates was 30 weeks with median birth weight of 1320gms. Slightly more than half of them delivered by normal vaginal delivery remaining by LSCS. APGAR score at 1 minute and 5 minutes was seven and nine respectively in 86.6% of babies. Among enrolled dyads, 8% had AEDF and 7 % had REDF perinatally. 77% of enrolled mothers had received antenatal steroid coverage, 15% of mothers had a history of PROM with 4% having h/o chorioamnionitis. Among all the enrolled babies 12 (4.9%) developed NEC, three babies developed severe disease. Mode of the delivery was predominately LSCS. Out of 12 cases, two thirds were male and remaining one third were females. Only one third of the cases had abnormal umbilical Doppler pattern. All the cases had good antenatal steroid coverage. Out of all cases, 80% of babies had respiratory distress. During the course of hospital stay two third of the babies had sepsis screen positive, out of which two babies had blood culture positivity. Forty percent of babies reacquired mechanical ventilatory support, 6% babies’ reacquired inotropic support during hospital stay. 2-FL levels in the breast milk were analysed by LC-MS/MS. Primary outcome was for to look for association between the levels of two -FL and NEC occurrence. We found that the 2-FL levels were less than 100µg/ml for NEC stages more than 2B, whereas the 2-FL levels in the respective controls were more than 200 µg/ml. The correlation between the levels of 2- FL in human breast milk between cases and controls was statistically significant with a P value of 0.03. In our study, there was also a significant association between disease occurrence and the APGAR at 1 minute (p-value 0.046), neonatal sepsis (p-value 0.001), neonatal shock (p-value 0.013), and the need for inotropic support (p-value 0.013). Multivariable logistic regression done using these factors and 2-FL as a predictive factor did not show statistically significant association between the levels of 2- FL and NEC occurrence. Our secondary aim was to see for any association between the levels of 2-FL and the stage of NEC according to the Bells staging. There was an inverse correlation between the levels of 2-FL and the stage of the disease with a correlation coefficient of -0.57. In our study, we also examined the predictive ability of 2-FL levels for the development of among newborn babies. Using 2-FL as the index test and NEC as an outcome variable, the ROC curve was generated. The area under the curve being 0.771 emphasises that 2-FL has a fair predictive value for the development of NEC. Discussion The underlying mechanisms through which 2-FL attenuates the occurrence of NEC are unclear, and only a handful of studies have explored this topic. In a study by Good et al. (7), the administration of 2-FL to neonatal rats resulted in a reduction in the development of NEC via upregulation of vasodilatory molecules, particularly endothelial nitric oxide synthase (eNOS), which restores intestinal perfusion. This demonstrates that 2-FL protects against NEC by improving mesenteric perfusion. Sodhi et al. also demonstrated that the inhibition of toll-like receptors (TLRs) is another mechanism by which 2-FL provides protection against NEC (7). Autran et al. (17) demonstrated in animal models that neonatal rats fed milk supplemented with 2-FL had lower pathological scores for NEC compared to those fed formula milk without HMOs. In our study, the median 2-FL levels in cases (135.54µg/mL) were significantly lower than those in controls (345.6µg/mL). This indicates that higher 2-FL levels are protective against the development of NEC. Our findings contrast with a study by Wejryd et al. (18), which showed that out of 15 HMOs in breast milk, a low level of the neutral oligosaccharide LNDH was associated with an increased risk of developing NEC. Moreover, their study failed to show any statistically significant association between 2-FL levels and the development of NEC. However, this lack of association might be attributed to insufficient statistical power (18). Secondary Outcome: Association between 2-FL Levels in Breast Milk and NEC Staging A study by Cilieborg et al. (20) in preterm piglets showed that the addition of 5 g/L of 2-FL to infant formula reduced both the incidence and severity of NEC. In our study, we examined the association between 2-FL levels and the severity of NEC, as represented by Bell’s staging. We found an inverse correlation between 2-FL levels and the stage of NEC, with a correlation coefficient of -0.57. The lowest 2-FL level observed in our study was 7.047 µg/mL, corresponding to stage 3B of the disease. This finding aligns with the study by Cilieborg et al. (20). Additionally, we assessed the predictive value of 2-FL levels below which NEC cases increased. We found that 2-FL levels below 311 µg/mL had a sensitivity of 80% and specificity of 60% for predicting disease occurrence. However, no human studies have been conducted to examine the predictive value of 2-FL levels. Based on the results of our study, we emphasize the importance of conducting trials to evaluate the effects of adding 2-FL and other HMOs to infant formula. Limited clinical data suggest that adding HMOs to infant formula is safe and offers acceptable tolerance and growth-promoting benefits (21). CONCLUDING REMARKS The study demonstrates an inverse relationship between the severity of NEC and the levels of 2-FL in human breast milk, emphasizing the role of 2-FL in mitigating disease occurrence. It highlights the need for well-designed studies to investigate the levels of 2-FL in human breast milk and its role in the prevention of NEC. REFERENCES 1.) Hackam DJ, Sodhi CP, Good M. New insights into necrotizing enterocolitis: From laboratory observation to personalized prevention and treatment. J Pediatr Surg. 2019 Mar;54(3):398-404. 2.) Alecia M. Thompson-Branch, Tomas Havranek; Influences of Feeding on Necrotizing Enterocolitis. Neoreviews November 2018; 19 (11): e664–e674. 3.) Alsaied A, Islam N, Thalib L. Global incidence of Necrotizing Enterocolitis: a systematic review and Meta-analysis. BMC Pediatr. 2020 Jul 13;20(1):344. 4.) Neu J. Necrotizing enterocolitis: the mystery goes on. Neonatology. 2014;106(4):289–295 5.) Rich BS, Dolgin SE. Necrotizing Enterocolitis. Pediatr Rev. 2017 Dec;38(12):552–9. Bering SB. 6.) Lucas A, Cole TJ. Breast milk and neonatal necrotising enterocolitis. Lancet. 1990 Dec 22-29;336(8730):1519-23. 7.) Ballard O, Morrow AL. Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am. 2013 Feb;60(1):49-74. 8.) HMO’ss to Prevent Gut Dysfunction and Necrotizing Enterocolitis in Preterm Neonates. Nutrients. 2018 Oct 8;10(10):1461. 9.) Bode L. The functional biology of HMO’ss. Early Hum Dev. 2015 Nov;91(11):619-22. 10.) Bode L. HMO’ss: every baby needs a sugar mama. Glycobiology. 2012 Sep;22(9):1147-62. doi: 10.1093/glycob/cws074. Epub 2012 Apr 18. PMID: 22513036 11.) Wiciński M, Sawicka E, Gębalski J, Kubiak K, Malinowski B. HMO’ss: Health Benefits, Potential Applications in Infant Formulas, and Pharmacology. Nutrients. 2020 Jan 20;12(1):266 12.) Ballard O, Morrow AL. Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am. 2013 Feb;60(1):49-74. 13.) Sodhi CP, Wipf P, Yamaguchi Y, Fulton WB, Kovler M, Niño DF, Zhou Q, Banfield E, Werts AD, Ladd MR, Buck RH, Goehring KC, Prindle T Jr, Wang S, Jia H, Lu P, Hackam DJ. The HMO’ss 2’-fucosyllactose and 6’-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling. Pediatr Res. 2021 Jan;89(1):91-101. 14.) Coppa GV, Pierani P, Zampini L, Carloni I, Carlucci A, Gabrielli O. Oligosaccharides in human milk during different phases of lactation. Acta Paediatr Suppl. 1999 Aug;88(430):89-94 15.) Meister AL, Doheny KK, Travagli RA. Necrotizing enterocolitis: It’s not all in the gut. Exp Biol Med (Maywood). 2020 Jan;245(2):85-95. 16.) Bode L. HMO’ss: every baby needs a sugar mama. Glycobiology. 2012 Sep;22(9):1147-62. doi: 10.1093/glycob/cws074. Epub 2012 Apr 18. PMID: 22513036 17.) Autran, C., Schoterman, M., Jantscher-Krenn, E., Kamerling, J., & Bode, L. (2016). Sialylated galacto-oligosaccharides and 2′-fucosyllactose reduce necrotising enterocolitis in neonatal rats. British Journal of Nutrition, 116(2), 18.) Wejryd E, Martí M, Marchini G, Werme A, Jonsson B, Landberg E, Abrahamsson TR. Low Diversity of HMO’ss is Associated with Necrotising Enterocolitis in Extremely Low Birth Weight Infants. Nutrients. 2018 Oct. 19). van der Heide, M., Mebius, M.J., Bos, A.F. et al. Hypoxic/ischemic hits predispose to necrotizing enterocolitis in (near) term infants with congenital heart disease: a case control study. BMC Pediatr 20, 553 (2020). 20.) Cilieborg MS, Jensen ML, Bering SB, et al. (2012) A milk oligosaccharide, 2’-fucosyllactose, may ameliorate necrotizing enterocolitis in preterm pigs. Breastfeeding Med 7, 571. 21) Vandenplas Y, Berger B, Carnielli VP, Ksiazyk J, Lagström H, Sanchez Luna M, Migacheva N, Mosselmans JM, Picaud JC, Possner M, Singhal A, Wabitsch M. HMO’ss: 2’-Fucosyllactose (2’-FL) and Lacto-N-Neotetraose (LNnT) in Infant Formula. Nutrients. AUTHORS’ CONTRIBUTION: SN: data collection; analysis of data; writing original draft; SVA: conceptualization (lead); supervision of data collection, analysis; finalizing original draft; SD: conceptualization (supporting); methodology; analysis of data; AP: sample processing; JK: supervision of data collection; analysis of data: writing original draft. 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Authors Affiliations Santosh Navi PGIMER View all articles by this author Savita Attri 0000-0002-1783-9094 [email protected] Post Graduate Institute of Medical Education and Research View all articles by this author Sourabh Dutta Post Graduate Institute of Medical Education and Research View all articles by this author Ajay Patial Post Graduate Institute of Medical Education and Research View all articles by this author Jayakanthan Doss Post Graduate Institute of Medical Education and Research View all articles by this author Metrics & Citations Metrics Article Usage 321 views 152 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Santosh Navi, Savita Attri, Sourabh Dutta, et al. 2-Fucosyllactose in human breast milk can predict the severity of necrotizing enterocolitis. Authorea . 17 January 2025. 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