Perinatal Environment Shapes Microbiota Colonization And Infant Growth: Impact On Host Response And Intestinal Function

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Perinatal factors, especially those associated with birth, including both mode and place of delivery are critical to shaping the infant gut microbiota with potential health consequences. Methods: Gut microbiota profile of 180 healthy infants (n=23 born at home and n=157 born in hospital, 41.7% via caesarean section [CS]) was analyzed by 16S rRNA gene sequencing at birth, seven days and one month of life. Breastfeeding habits, infant clinical data, including length, weight and antibiotic exposure, were collected up to 18 months of life. Long-term personalized in vitro models of the intestinal epithelium and innate immune system were used to assess the link between gut microbiota composition, intestinal function and immune response. Results: Microbiota profiles were shaped by the place and mode of delivery, and they had a distinct biological impact on the immune response and intestinal function in epithelial/immune cell models. Bacteroidetes and Bifidobacterium genus were decreased in C-section infants, who showed higher z-scores BMI and W/L during the first 18 months of life. Intestinal simulated epithelium had a stronger epithelial barrier function and intestinal maturation, alongside a higher immunological response (TLR4 route activation and pro-inflammatory cytokine release), when exposed to home-birth fecal supernatants, compared with CS. Distinct host response could be associated with different microbiota profiles. Conclusions: Mode and place of birth influence the neonatal gut microbiota, likely shaping its interplay with the host through the maturation of the intestinal epithelium, regulation of the intestinal epithelial barrier and control of the innate immune system during early life, which can affect the phenotypic responses linked to metabolic processes in infants. General Microbiology Microbiota environment mode of birth antibiotics epithelial barrier immune system early programming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Microbial colonization plays an important role in numerous functions, including digestion, metabolic reactions and trophic effects, and it also influences the development and maturation of the host’s innate and adaptive immune system [1–3]. The mode of birth is a key factor shaping early microbial colonization [4–6]. Vaginally born (VAG) infants acquire microbial communities resembling the maternal vaginal and gut microbiota, whereas infants born via C-section acquire environmental-like bacteria such as Staphylococcus, Corynebacterium and Propionibacterium spp. [7]. In addition, CSs are associated with lower microbial diversity, delayed colonization of Bacteroides and Bifidobacterium spp. and reduced immune responses [8]. The CS rate increased by an annual increment of 3.7% from 2000 to 2015 worldwide [9,10]. In Europe, the average CS rate is 28% [11], although the World Health Organization (WHO) recommends a rate of 10–15% [12]. Epidemiological studies have linked CSs with a higher risk of non-communicable diseases such as obesity [13,14] and allergy [15]. Indeed, the CS procedure is characterized by pre- and intra-partum antibiotic exposure and other medical practices, which may affect early gut colonization and predispose the infant to developing immune-related disorders later in life, including asthma [16–18], allergy [19–21], obesity [22,23] and diabetes [24,25]. Hospital interventions during birth are critical for pioneer microbial colonizers and proper immune system maturation [26,27], which may impact adult health [28,29]. However, neonatal microbiota colonization in the absence of hospital interventions remains underexplored. Furthermore, the hospital environment (high-level disinfection and antibiotic therapy) has an impact on microbial exposure, thereby extending the hygiene hypothesis to the time of birth [30,31]. Home births (HBs) increased by 77% between 2004 and 2017 in the United States, while the rate of birth-center deliveries doubled during the same period [32]; however, in Europe, such births account for less than 1% of all deliveries, except in the Netherlands, where HBs represent 16.3% of births [33]. Recently, a distinct microbiota profile has been reported in VAG neonates born at home or in hospital [34], although the impact on microbiota development and the potential effects on neonatal health are not fully understood. In this study, we investigate the influence of birth-related factors, including the place and mode of delivery on early colonization during the first month of life and on infant growth during the first 18 months. Furthermore, to understand the potential biological mechanisms involved, in vitro gut models are used to study the impact of distinct microbiota patterns on intestinal function and innate immune system maturation. Results Study population No differences were observed in the neonatal weight between groups, which showed a median of 3250 g (range 2973–3573 g). Other maternal clinical parameters are presented in the supplementary material (Additional file 1). Despite all the babies were born at full term, the CS-born neonates were born before the both groups of vaginally delivered neonates (39 weeks of pregnancy for CS births and 40 weeks for the VAG and HB neonates, respectively) ( p=0.004 ). The HB neonates showed higher length measurements than the hospital-born infants for both delivery modes ( p=0.003 ). Additionally, the HB infants had higher ratios of exclusive breastfeeding than the hospital-delivered infants at both seven and 31 days of life ( p<0.001 ). Perinatal factors related to the place and mode of delivery shape the neonatal microbiome composition at birth At birth, the place (hospital versus home) was the main contributor of neonatal microbiota composition ( p=0.001 ), followed by the mode of birth ( p=0.025 ) (Figure, 1A). Other perinatal factors did not significantly influence the neonatal microbiota during delivery. The place and mode of birth shaped the microbial richness and diversity at amplicon sequence variant (ASV) level (Additional file 2). The hospital-born neonates showed a bacterial community with greater richness ( p=0.002 ) and diversity ( p=0.072 ) at birth than the HB neonates. The CS-born neonates harbored higher index of observed species ( p=0.023 ), diversity ( p=0.001 ) and richness ( p=0.031 ) than the HB infants. The neonatal fecal microbiota was dominated by the Proteobacteria phylum, followed by Firmicutes and Actinobacteria (Additional file 3). The hospital-born infants showed higher relative abundances of the Finegoldia ( p<0.001 ), Clostridioides ( p<0.001 ), Klebsiella ( p=0.025 ) and Peptoniphilus ( p=0.006 ) genera, including Clostridioides difficile ( p<0.001 ) and Clostridium neonatale ( p=0.006 ), while the HB infants showed higher relative abundances of the Staphylococcus ( p<0.007 ) and Enterococcus ( p<0.001 ) genera. As for the mode of birth, the VAG infants showed higher relative abundances of the Bacteroidetes ( p=0.035 ) and Firmicutes ( p=0.035 ) phyla, including the Bacteroides and Escherichia/Shigella genera. A linear discriminant analysis of effect size (LEfSe) was used to confirm which genera were responsible for the clustering of the rectal swab microbial population (Additional file 4). The microbiota of the HB neonates was enriched by species from the Enterococcus and Staphylococcus genera, while the hospital-born infants were enriched with species from the Klebsiella , Veillonella and Clostridioides genus. We also observed a microbial core at each time point and noted some unique genera not shared between the groups (Additional file 5). For instance, we observed that the Akkermansia genus was only present in the HB. Perinatal factors related to the place and mode of delivery shape neonatal microbiota development Mode of birth ( p=0.001 ) and time ( p=0.001 ) were the main contributors to the overall microbiota structure during the first month of life at ASV level. Generally, neonatal microbiota at birth showed significantly higher microbial richness (Chao1 index) and diversity (Shannon index) than the microbiota at 7 and 31 days of life (Additional file 2). No differences in alpha diversity (Chao 1, Shannon index) were found according to the place or mode of delivery at the 7- or 31-day time points. Distinct colonization patterns were identified between the hospital born (VAG and CS) and HB neonates (Figure, 1D). The mode of birth shaped the microbiota colonization process at 7 (Adonis p<0.001 ) and 31 days (Adonis p<0.005 ) at ASV level. The infant microbiota showed differing development in the VAG and CS-delivered neonates at the phylum and family levels (Figure, 1D). The VAG deliveries exhibited a colonization pattern somewhere between the patterns of the CS-born and HB deliveries. The HB and CS-born infants showed significant differences in the Firmicutes phylum at delivery ( p=0.035 ) and in the Proteobacteria at seven days ( p=0.019 ), although they did not show differences when compared to the hospital-based VAG infants. The relative abundance of the Actinobacteria phylum increased during neonatal life in the both groups of vaginally (VAG and HB) born infants but not in the CS-born neonates (Additional file 3). In the HB neonates, the Actinobacteria increase occurred from delivery to 31 days, while in the hospital-based VAG neonates, the increase was delayed from seven to 31 days of life. The Bacteroidetes phylum abundance was slightly higher in the vaginal births (VAG and HB) at seven ( p=0.014 ) and 31 days ( p=0.01 ) than in the CS births. At the phylum level, the vaginal births (VAG and HB) showed higher relative abundances of the Actinobacteria ( p=0.001 ) and Bacteroidetes ( p=0.018 ) phyla, especially Bifidobacterium (p=0.003 at seven days), when compared with the CS-born infants at both times (7and 31 days). The CS-born infants were enriched with the Firmicutes phylum ( p=0.020 ), including Enterococcus ( p=0.005 at seven days) and Clostridium ( p=0.039 ). The HB neonates harbored a higher relative abundance of Actinobacteria ( p=0.004 and p=0.006 at 7 and 31 days, respectively) and a lower relative abundance of Proteobacteria ( p=0.026 and p=0.008 at 7 and 31 days, respectively) when compared with the hospital-born infants (VAG and CS) at both times. Hospital-born infants had higher relative abundances of the Klebsiella species and lower relative abundances of Bifidobacterium genus, including B. bifidum ( p=0.013 ), and Collinsella genus, including Collinsella aerofaciens ( p=0.028 at 31 days), compared with the HB neonates. In the vaginal births (both, VAG and HB), the relative abundances of the Collinsella and Bacteroides genera increased during the first month of life. An opposite trend was observed for the Escherichia and Enterococcus genera (Figure 2). In the CS-born infants, Escherichia , Enterococcus and Klebsiella genera increased from birth to seven and 31 days (Figure 2). The relative abundance of Bifidobacterium genus was higher in the vaginal births, especially the HB infants at 7 ( p<0.001 ) and 31 days ( p=0.004 ), when compared with the CS births. Moreover, CS-neonates harbored a higher relative abundance of the Clostridium sensu stricto genus than the HB infants at 7 days ( p=0.007 ), although no difference was observed with the hospital-based VAG infants ( p=0.250 ), indicating intermediate colonization patterns in neonates born in hospital via vaginal route. At both 7 and 31 days, the Bacteroides genus was present between the vaginal-delivered neonates but not the CS-delivered infants, who showed Enterobacter as a unique genus at 31 days. In a subset of samples, total bacterial counts by qPCR were significantly lower at birth than the counts obtained at seven days in all three groups (Additional file 6). We also found that the CS-born fecal samples showed significantly lower bacterial counts than the VAG ( p=0.043 ) and HB ( p=0.008 ) samples at delivery. The HB infants showed higher total bacterial counts than the VAG ( p=0.008 ) and CS-born ( p=0.043 ) infants at seven days. Similarly, in the case of the Bifidobacterium genus counts in the vaginal-delivered groups (HB [ p<0.001 ] and VAG [ p=0.003 ]), the counts increased during the first month. Furthermore, the HB infants had higher Bifidobacterium counts than the VAG and CS-born infants at seven ( p=<0.001 for CS, p=0.004 for VAG) and 31 days ( p=0.027 for CS, p=<0.001 for VAG). Impact of the perinatal environment on the infant weight status at 18 months Higher BMI and W/L z-scores were observed in the CS-born infants than in the HB ( p=< 0.001 for BMI, p< 0.001 for W/L at 12 months) and hospital-born vaginal births ( p=< 0.001 for BMI and p= 0.003 for W/L at 12 months) (Figure, 3 A-B). Indeed, at 18 months of life, CS infants exhibit also higher BMI z-scores than HB ( p< 0.001) and VAG (p= 0.016) children. Additionally, a multivariate linear analysis (adjusted by breastfeeding duration, antibiotic intake during the first year of life, maternal pre-gestational BMI, and BMI and W/L z-scores at delivery) showed the CS-born neonates to exhibit significantly higher BMI and W/L z-scores across the first 18 months of life. Microbiota predicted functionality during the first month of life is influenced by the birth mode and place The inferred microbial functionality at birth was mainly affected by the mode of birth ( p<0.001 ), but also by the birthplace ( p=0.049 ). In addition, mode of birth also influenced the microbiota predicted functionality at 7 ( p=0.001 ) and 31 days ( p=0.02 ); however, the birthplace was only significant at 7 days ( p=0.001 ) (Figure 4, A-B). Regarding concrete functions, the microbiota of the vaginal-delivered infants (VAG and HB) were enriched in functional routes related to the synthesis of secondary metabolites ( p<0.001 ), amino acids ( p=0.002 ), lipids ( p=0.034 ) and carbohydrate metabolism ( p=0.046 ) when compared with the CS-born neonates at delivery (Additional file 7). At seven and 31 days, the vaginal-delivered infants showed microbiota enriched in lysine, phenylalanine, tyrosine, tryptophan, valine, leucine and isoleucine biosynthesis pathways. At one month of life, the CS-born infants expressed higher energy metabolism pathways, including carbohydrates, lipids, and propanoate biosynthesis. The immune-system-related paths (e.g. antigen processing and presentation [ p<0.001 ] and NOD-like receptors [ p<0.001 ]) were overrepresented in the CS-born infants at birth. Contrarily, the lipopolysaccharide (LPS) biosynthesis routes were enriched in the vaginal-delivered infants at all time points (Figure 4, C-D), being also influenced by the birthplace. The genera that mostly contribute to the differences in these predicted routs were Escherichia/Shigella , Klebsiella and Clostridium sensu stricto . However, the CS-delivered infants showed increasing representation of pathways related to bacterial toxins at seven ( p=0.011 ) and 31 days ( p=0.004 ) when compared with the HB neonates. Fecal supernatants induce the mRNA expression of TLR4 and IRAK4 in intestinal epithelial cells The HT-29 reporter cells showed increased NF-kB activation independently of the place and mode of delivery in all the groups (data not shown). All fecal supernatants induced IL8 and TNF-a production in the HT-29 cells when compared with control cells exposed to cell culture media (Figure 5, A). No differences were found between the groups in relation to any of the cytokines. HB fecal supernatants up-regulated the TLR4 mRNA in a 1.405-fold change ( p=0.024 ) and the IRAK4 mRNA in a 2.654-fold change ( p<0.001 ) when compared with the control samples. This increase was not observed in the cells exposed to hospital-born (VAG and CS) neonatal samples (Additional file 8). Generally, the fecal supernatants all reduced the mRNA expression of the tight-junction proteins, including zonulin-1 ( HP ) (0.202- and 0.265-fold change in the HB and CS samples, respectively), e-cadherin ( CDH1 ) (0.226- and 0.326-fold change in the HB and CS, respectively) and occludin ( OCLN ) (0.192- and 0.253-fold change in the HB and CS). However, no differences were found between the different studied groups. HB fecal supernatants trigger higher immune response in macrophage-like cells than CS fecal supernatant. The mode of birth significantly affected the production of cytokines in the PMA-differentiated THP-1 cells (Figure 5, B). The HB and VAG fecal supernatants had a higher pro-inflammatory capacity than the CS samples. Higher levels of IL6 and IL8 were detected after HB ( p=<0.001 ), followed by the VAG ( p=0.043 ) samples, when compared with the CS-born fecal supernatant. The VAG and HB samples triggered a significantly higher response than the control condition for IL6, IL8 and TNF-a, which was not observed in cells exposed to CS samples. The CS fecal supernatants down-regulated the TLR4 (0.509-fold change, p=0.006 ) and FOS mRNA expression (0.238-fold change, p=0.038 ) (Additional file 8). The expression levels were not quantifiable for the interferon gamma ( IFN- g ) and IL10 genes in either the HT-29 or THP-1 cell lines following faecal supernatant exposure. Triple co-culture system for host–microbiome interaction Place and mode of birth impacts on intestinal barrier function and maturation With regard to the integrity of the cell monolayer (Additional file 9), HB promoted a higher increase in the transepithelial electrical resistance (TEER) values after seven days of exposure (Figure 6, A-B) when compared with hospital birth (VAG [ p<0.001 ] and CS [ p<0.001 ]). These results were confirmed via the measurement of lucifer yellow dye (LY) transport through the epithelial layer. Despite all the fecal supernatants triggered an increase in LY transport when compared to control condition ( p<0.05 ), hospital-based birth, both VAG (2.1·10 -6 ±5.7·10 -7 cm/s, p=0.018 ) and CS (2·10 -6 ±3.5·10 -7 cm/s, p=0.033 ), led to higher LY transport than HB (1.5·10 -6 ±1.7·10 -7 cm/s). The intestinal alkaline phosphatase (IAP) activity was also measured in the supernatant of cell cultures from both; the apical and basal compartments (Figure 6, D). Indicative of functional cell polarization, the IAP activity was significantly higher ( p=0.006 ) in the apical compartment at 7 days of treatment, while all the fecal supernatants enhanced the IAP activity when compared with the control. Similar to the TEER and LY results, HB samples induced higher IAP activity than the ones observed in VAG ( p=0.043 ) and CS samples ( p=0.049 ). Furthermore, higher mucus production was observed in cells exposed to hospital fecal samples (0.92±0.2 mg/ml) when compared with the HB samples (0.69±0.09 mg/ml) ( p=0.006 ). Immune system response The immune response to the fecal supernatants was generally higher in the basal than in the apical compartment. In the simulated intestinal epithelium, the exposure to CS-born fecal supernatants down-regulated (0.43-fold change) the IRAK4 mRNA expression when compared with the control-no stimulus ( p=0.01 ) and HB samples (0.45-fold change, p=0.001 ). Contrarily, the CS-born fecal supernatants up-regulated the toll-interacting protein ( TOLLIP) mRNA ( p=0.007 ) expression in the HB samples (1.54-fold change) (Table 1). Table 1 . Gene expression of cells in the apical and basal compartment in the triple co-culture system after 7 days of exposure HB CS Fold expression p-value Fold expression p-value Apical compartment (Caco-2, LSTH-17 cells) HP 1.139 (0.767 - 1.627) 0.495 1.097 (0.957- 1.214) 0.106 CDH1 1.054 (0.462 - 1.675) 0.841 0.916 (0.736-1.237) 0.399 OCLN 1.163 (0.673 - 1.534) 0.531 1.321 (0.867-1.702) 0.083 IRAK4 (#) 0.94 (0.610 - 1.168) 0.837 0.425 (0.273-0.595) 0.010* ↓ TLR2 1.492 (0.837 - 2.142) 0.181 1.302 (0.960-1.710) 0.061 TLR4 0.89 (0.722 - 1.074) 0.201 0.826 (0.482-1.276) 0.475 TOLLIP 0 (0.000 - 1.381) 0.509 1.231 (0.658-2.076) 0.474 Basal compartment (THP-1 cells) TLR4 3.566 (1.103 - 13.629) 0.094 1.117 (0.235 - 7.013) 0.896 TLR3 1.346 (0.939 - 1.853) 0.378 3.546 (1.712 - 8.099) 0.094 TOLLIP 5.182 (3.667 - 9.351) <0 .001*↑ 7.186 (4.388 - 11.727) 0.010*↑ IL10 7.888 (3.496 - 31.769) < 0.001*↑ 1.737 (0.912 - 5.478) 0.061 Total RNA was extracted from cells treated with homebirth (HB) samples and C-section (CS) fecal supernatant. For each condition, n=6 in triplicates, data was presented as fold change expression (95% C.I). Values expressed relative expression fold-change of each condition compared to control. P<0.05 (*) and blond letters marked significant differences between treatment and control, up (↑) or down (↓) regulation was represented by the arrows. Symbol # represented genes expression that was different between HB and CS. IL8 was detectable at all time points in the apical compartment (Figure 6, E). Generally, the IL8 exhibited a gradual increase until the fifth day of exposure (126±39% average increase for the three groups). Thereafter, the IL8 levels decreased until the end of treatment (7 days, 99±24% average of three groups). The IL6 concentration was only above the detection limit at the final time point (7days), and there were no differences between the groups. In the basal compartment, similar to the epithelium-like layer, we observed a 7.2-fold change in the down-regulation of the TOLLIP mRNA expression after exposure to CS-born samples ( p<0.001 ) (Table 1). IL8 release was observed at 24 hours (151.3±42.7% increase from the control), with the maximum being seen after five days of treatment (164±65% increase). At day seven, the IL8 levels reached similar values to those seen on the first day of treatment (82±67% increase) (Figure 6, F-J). The IL6 production by the THP-1 cells showed a late response to faecal supernatant exposure, lasting from day one of treatment until day three (218±223% increase from control); however, the increase was only significant for the HB ( p=0.003 ) and VAG ( p=0.017 ) samples. At the subsequent time points, the IL6 release was stabilised to concentrations similar to those seen on day one (61.45±11.69% for CS, 48.88±11.14% and 59.97±13.4% for HB). No significant differences were observed between the groups in terms of the cytokine production patterns over the seven days. Discussion In this study, we evaluated gut microbiota evolution during the first month of life in CS-delivered and both HB and hospital-based VAG infants. Our results highlight the relevance of perinatal factors to the gut microbial colonization pattern, which affects the innate defensive mechanisms and functional maturation at the intestinal level. CS procedures are associated with specific conditions, including the use of antibiotics, longer hospitalization, low neonatal–maternal contact and the delayed initiation of breastfeeding [35,36], which may influence microbial colonization patterns [26]. A higher percentage of bottle or mixed feeding is commonly observed in CS-born infants [35], including the studied cohort. In our cohort, the HB infants showed a higher rate of exclusive breastfeeding than the hospital-born infants (95% in HB versus 67% and 61% in CS and VAG at hospital, respectively). Furthermore, the practices associated with hospital delivery, as antibiotic use, vaginal cleansing, controlled maternal food intake and mobility, oxytocin administration or anesthesia, may affect the maternal microbiota and, consequently, mother–infant microbial transmission. Those factors together with prematurity would have a relevant impact on infant development that remains poorly understood. Additionally, these birth-related factors may affect the microbial establishment process and cannot be independently studied. Therefore, our results sum up the consequences of all the concomitant factors affecting the different groups of study. The hospital environment, especially in the case of CSs, can be considered highly intervened condition characterized by the high pressure of antibacterial therapy and instrumentalization. HB rates have increased in Europe over recent decades, ranging from 0.1% in Sweden to almost 20% in the Netherlands [37]. However, limited information concerning the possible benefits of HB is currently available due to the lack of adequate randomized clinical trials meaning that it is not possible to determine the risk of neonatal–maternal mortality and morbidity during HB [38]. Further, little is known about the impact of a non-hospital environment on infant gut colonization. We observed that the mode and place (hospital versus home) of birth shaped the neonatal microbiota. The microbiota of hospital-delivered infants was enriched with gram-positive anaerobic cocci, including the Peptoniphilus and Finegoldia genera, while those of HB infants exhibit higher relative abundances of species from the Enterococcus and Bifidobacterium genera. In all three groups, time was the main factor affecting the composition of the infant microbiota, with a significantly different pattern between groups observed at birth, but not at the subsequent time points. Primary events concerning gut microbial colonisation may impact the assembly and acquisition of the early microbiome, likely having long-lasting consequences for gut ecology [29]. At delivery, we observed higher diversity indices in the neonatal microbiota than at the subsequent time points; however, the quantitative total bacteria count was lower at delivery. This is likely caused by bias in the sequencing data obtained from low biomass samples and by higher contact with environmental contaminants. The HB infants had lower microbial diversity but a higher total number of bacteria than the hospital-born infants, likely due to the latter having increased contact with bacterial environmental contaminants [6]. We found clearly identifiable differences in the global microbiota structure during time between the three groups, even at the phylum level, which is in agreement with previous studies [4,6,34]. Interestingly, the differences in the colonization patterns become more pronounced over time, at least during the first month of life, indicating the importance of birth-related events to neonatal colonization processes, which may affect the microbiome composition in later developmental stages. Altered microbial colonization has been also observed in preterm infants compared to term infants, and these microbial shifts have been mainly linked to antibiotics exposure, caesarean section, hospitalization, use of formula feeding. Regarding term infants, some studies have shown shifts related to delivery mode in term infant microbiota up to 3 months post-delivery [8,39], other authors observed no differences in terms of microbiota composition in neonates born by CS beyond the first days of life [40]. Thus, the duration of the shifts in infant microbiota associated to delivery mode has reported contradictory results and needs to be further evaluated. Similar to prior studies, we found that CS-born infants showed higher relative abundances of Clostridium and Klebsiella and lower species from the Bifidobacterium genus [6]. We observed that both VAG and HB infants had higher number of Bifidobacterium species than CS-born neonates. The depletion of the Bifidobacterium species in the gut environment has been associated to immune-related diseases [41,42], and members of this genus are commonly used as probiotics due to their capacity to modulate microbiota–immune system homeostasis [43]. Could these shifts in the microbiota composition alter the functional profile of the neonatal microbiota? Descriptive and observational studies are relevant to understanding microbiome evolution during the neonatal period; however, mechanistic studies of the host–microbiome interplay during early life are still required. Thus, microbiota functional analysis has been proposed as a tool for clarifying the host–microbiome interactions [44]. Our results of the predicted metagenome from 16S rRNA sequencing data showed functional differences between the place and mode of birth in the infant microbiota at delivery, 7 and 31 days. Several amino acid (AA) biosynthesis routes were over-represented in the microbiota from vaginal-delivered infants when compared with the CS-born infants, including tryptophan-related paths. It is known that AAs serve as regulators of several metabolic pathways in the host [45] and more specifically, tryptophan interacts with the immune system through microbial serotonin production [46], regulation of TLRs or interacting with the aryl hydrocarbon receptor–microbiota–immune system path [47,48]. We also found that vaginal-delivered infants, especially HB babies, had a microbiota enriched with LPS biosynthesis-related functions. Similarly, Wampach et al. found differences in the earliest functional profile according to the delivery mode, including LPS biosynthesis routes being enriched in vaginal deliveries when compared with CS-born neonates [49], which may influence immune system maturation and neonatal health. Despite these observations, these results need to be further evaluated since the prediction of microbiota functionality has been shown to report results that may be not conclusive [50]. Do these differences in the microbiota influence the host immune system response? Despite studies in animal models highlighting the possible effects of microbial colonization patterns on the host gut epithelium maturation and immune system response, little evidence is available from human studies. To the best of our knowledge, this is among the first studies to address this important issue. The intestinal epithelium is the gateway through which gut microbiome–host crosstalk effects intestinal functionality in the form of enterocytes maturation, mucus production or epithelial barrier development, being a key anatomical location for host–microbiome interplay. The samples from HB infants exhibited a higher immune stimulatory capacity than those from hospital-born infants (both VAG and CS), with an increased ability to induce the expression of immune system-related genes ( TLR4 and IRAK mRNA) and cytokine responses in the HT-29 and THP-1 models, including IL6 and IL8. In concordance with our results, Wampach et al. identified the higher immunostimulatory potential of the microbiota of vaginal-delivered infants when compared with CS-born infants, although they used LPS purified from infant fecal samples and primary human macrophages differentiated into dendritic cells [49]. Combellick et al. noted the higher expression of TLR4 and IL8 mRNA by the HT-29 cell line following exposure to sterile fecal supernatant from HB infants when compared with hospital-born infants. However, they also found mRNA upregulation of the anti-inflammatory cytokine TGF- b in hospital-born neonates [34]. We observed that the THP-1 cell line response was more affected by the mode and place of delivery than the HT-29 cell line, which highlighted the importance of the epithelial integrity and the innate immune system on the in vitro assessment of the host-microbiome interplay. Most prior studies with similar objectives involved acute exposure on unique cell lines. We hypothesized that acute exposure to microbial products could not accurately reflect the biological effect of microbial metabolites and so proposed long-term (7-days) in vitro exposure assays, including the crosstalk between different cell types, which enabled us to obtain personalized results for each participant, thereby translating the individual signatures to the in vitro system. In our model, the HB fecal supernatants induced higher gut barrier integrity (Figure 5) and functionality (IAP; Figure 5) following a time-dependent response that highlighted the relevance of the dynamics of host–microbiome interplay. Interestingly, the impaired closure of gut mucosal membranes has been shown, alongside higher intestinal permeability, in preterm infants who received formula feeding rather than breastfeeding [51]. This increased permeability could be related to allergic diseases in non-breastfed children [52] and other health disorders [53]. In addition, the HB samples induced the expression of anti-inflammatory molecules (e.g. IL10 , TOLLIP ) to a higher extent than the CS samples, indicating negative feedback on the inflammatory signaling in the gut. Specifically, IL10 down-regulate the microbiota-activated mucosal inflammatory cytokines, reinforce the gut epithelium barrier and control gut permeability, all essential factors to maintaining intestinal homeostasis [54]. Another key element of the innate immune response of the gut is the protective mucus layer covering the epithelium [55]. Higher mucus production was observed after cell-exposure to fecal supernatants obtained from hospital-born infants when compared with HB infants. Both, microbiota [56] and TLR expression [57] are involved in the regulation of mucus production. Despite lower mucus production in the gut being associated with disease phenotypes (e.g. inflammatory bowel disease, higher susceptibility to bacterial infections) [58] in adults, little is known about the role of mucins in neonatal colonization processes. We hypothesize that a penetrable mucus layer in newborns would allow for microbial colonization and interaction with the epithelium during the immune-priming window. Many researchers have discussed the possible relationship between CS and altered immune system development [26]. Generally, CS delivery is associated with the poor stimulation of the immune system [59–61]. Some researchers have proposed non-diverse environments in early life, including delivery, to play a role in immune maturation and triggering. Furthermore, recent evidence has shown that early exposure to rural areas or farm environments could affect microbial composition and diversity [62], which may be linked to a reduced risk of suffering atopies in adulthood [63]. Kirjavainen et al. recently described how a farm-like indoor microbiota could also decrease the asthma risk in a non-farm environment [64]. However, most of these results were derived from observational studies, as very few mechanistic analyses have been conducted to date. Our results suggest a possible link between CS and the delayed maturation of intestinal function and the innate immune system. Such a link could play a significant role in the diseases associated with intervention-based deliveries, including autoimmunity, allergy and other immune- and metabolic-related disorders. In this regard, CS-born infants from our cohort showed higher BMI and W/L z-scores during the first 18 months of life. Other researchers have reported similar results, associating CS with the risk of overweight in children [65,66]. These results could indicate the relevance of priority events in infant health, including those altering microbial colonization, , thereby supporting the early programming hypothesis [67,68]. Researchers have described how antibiotic therapy during early life modulates weight gain in different ways depending on the antibiotic dose in both animal [69] and human epidemiological studies [66]. It has been suggested that high-dose antibiotics can cause important reductions in the microbiota population, which may be related to the weight loss observed in some studies [70]. However, lower antibiotic doses would cause microbiota composition shifts, more than population size variation, and trigger the weight gain shown in the above-mentioned studies. Thus, it remains to be discovered whether the proposed mechanism could be extended to other perinatal factors that also disrupt the microbiota composition and transmission. The limitations of this study include the low number of participants and the possible confounding factors not included in the analysis (e.g. maternal diet, lifestyle or number of siblings, pets among others). Our microbiota analysis was based on the taxonomic profile obtained via 16S rRNA gene sequencing, which offers less resolution than complete shotgun metagenome sequencing. As we used sterile fecal supernatant, we observed the effects of soluble bacterial metabolites and also, of non-bacteria-related products, including growth factors or eukaryotic extracellular vesicles, which may have influenced the observed results. The use of cell lines may hamper the translational results, although it offers a reproducible and economically viable strategy for further testing on more physiologically relevant models. Among the strengths of the study are the inclusion of three groups and the comparison of CS and vaginal delivery at both hospital and home, including 18 months of follow up. We performed the cellular exposure assays in cellular models with different degrees of complexity and different exposure times, including the epithelial barrier function and maturation as relevant targets, together with the innate immune response. Yet, fecal supernatants contain a complex array of molecules representative of the in vivo condition, which retain inter-individual differences and features. Conclusion Our results may provide a mechanistic linkage between studies associating CS and immune-related diseases with colonization pattern alterations, although we cannot rule out other possible factors that might participate in the process. The study has shed light on the effects of hospitalization and HB on neonatal microbial colonization and on the possible effect on innate immune system development, specifically at the intestinal level. The results highlight both the importance of host–microbial contact during the first month of life and the dynamism of the process. However, further research is needed to determine the impact of these observations in neonatal in vivo clinical conditions. Such knowledge would facilitate the design of strategies for adjusting medical practices with the aim of reducing intervention during the birth process and ensuring the correct initiation of bacterial colonization and, consequently, the immune system response during early life. Methods Subjects and Sampling A prospective cohort study was conducted to compare the intestinal microbiota of delivered infants born at the hospital (VAG, n=92 and CS, n=65) and at home (HB, n=24). Infants with available biological samples at birth, 7 days and 1 month, together with clinical data, were included. Ethical approval for the study was obtained from the Ethics/Bioethics Committee for Clinical Research of Hospital La Fe, Hospital Clinic, Parc de Salut MAR and CSIC (Consejo Superior de Investigaciones Científicas) [ClinicalTrial.gov NCT03552939]. Pregnancy, intrapartum variables and anthropometric data were recorded (Additional file 1). Maternal age, maternal pre-pregnancy weight, weight gain over the pregnancy, maternal smoking status, mode of delivery, place of birth (home or hospital), birth weight and length, sex of the neonate, birth instrumentalization, maternal antibiotic exposure during pregnancy and maternal/infant antibiotic use at birth were also collected. Infant length and weight were also registered at birth, 1, 6, 12, and 18 months. Z-scores of anthropometric measures were electronically computed using WHO Anthro software (www.who.int/ childgrowth/software/en/) . The WHO Child Growth Standards provide child growth measures standardized by age and sex using z-score. Fecal DNA Extraction Total DNA was extracted from the fecal material (approx. 50-100 mg) using the Master-Pure DNA extraction Kit (Epicentre, Madison, WI, US) following the manufacturer’s instructions with the following modifications: samples were treated with lysozyme (20 mg/mL) and mutanolysin (5U/mL) for 60 min at 37ºC and a preliminary step of cell disruption with 3-μm diameter glass beads during 1 min at 6 m/s by a bead beater FastPrep 24-5G Homogenizer (MP Biomedicals). Purification of the DNA was performed using DNA Purification Kit (Macherey-Nagel, Duren, Germany) according to manufacturer’s instructions. DNA concentration was measured using Qubit® 2.0 Fluorometer (Life Technology, Carlsbad, CA, US) for further analysis. Sequencing and Bioinformatics Analysis Quality-trimmed and filtering was assessed using DADA2 v. 1.12.1 pipeline [74]. After quality examination, reads were trimmed at the 270th and 210th nucleotide in forward and reverse position, respectively. Additionally, adapters were also removed in the filtering process and a maximum of 2 expected errors was considered. The following denoising and merging steps were performed, and chimeras were also removed. Taxonomic assignment was conducted using the Silva v132 database with the addition of the specie level classification by the same database. Taxa occurring <3 reads in at least 10% of the total samples number and those representing less than 0.01% of the reads across all the samples were filtered. Furthermore, the decontam package v. 1.4.0 [75] in R environment [76,77] was used to determine the presence of potential contaminants-related sequence. Samples with less than 1000 reads were also removed from the final analysis (n=6). One of those samples was from a vaginally born infant with samples only at delivery time and this infant data was eliminated in further analysis. The final ASV (amplicon sequence variance) table is listed in Additional file 11 . The 16S rRNA gene sequence data generated is available through NCBI Sequence Read Archive Database under project accession number BioProject ID PRJNA614975. Predictive inferred functional analysis was performed using PICRUST v. 1.1.4 pipeline [78] and the linear discriminant analysis effect sized (LEfSe) analysis was performed for the biomarker discovery using a size-effect cut-off of 3.0 on the logarithmic LDA score [79] using the R code available in the yingtools2 package ( https://rdrr.io/github/ying14/yingtools2/man/lefse.html ) and the Dr. Huttenhower’s lab galaxy repository of bioinformatic tools. Bacterial Quantification by quantitative PCR Analysis A small subset of samples (n= 248) according to DNA availability (delivery n=78; 7d n=100; and 31d n=86) were used for the specific bacterial count determination by the qPCR. Total bacterial and Bifidobacterium genus counts were measured by quantitative system based on the amplification of specific 16S rRNA gene region by use of Light Cycler 480 Real-Time PCR System (Roche, Basilea, Switzerland). The (Details in Additional file 11). Reaction mixture consisted in SYBR Green I master mix (Roche, Basilea, Switzerland), 0.25 µM of each specific primer set and 1 ul of DNA. The amplification process consists of one cycle at 95ºC for 5 min, followed by 40 cycles at 95ºC for 20 s, annealing temperature ( Additional file 12 ) for 10 s and 72ºC for 10 s. Melting curves were also assessed to test the specificity of the reaction. Standard curves for the specific targeted bacterial group were generated using Ct values and the calculated gene copies numbers were determined based on the fragment amplification length. Cell culture All the reagents for cell culture were purchased from Sigma-Aldrich, Spain, otherwise stated. NF-κB-SEAP HT29 reporter cells . The HT-29-transfected cell line was previously established in the Laboratory of Lactic Acid Bacteria and Probiotics of IATA-CSIC, by stable transfection of HT-29 cells with a NF-κB- secreted alkaline phosphatase (SEAP) plasmid (pNiFty2-SEAP; Invitrogen, Carlsbad, CA, US) as a reporter. Cell maintenance was performed in 75 cm 2 flask with Dulbecco's Modified Eagle Medium (DMEM) High glucose medium supplemented with 1% (v/v) L-Glutamine 200 mM, 1% (v/v) Na-Pyruvate, 1% (v/v) penicillin/streptomycin and 10% (v/v) of inactivated Fetal Bovine Serum (Biowest). Zeocin (200 µg/ml) (InvivoGen) was added to the medium for the clone selection in each passage. All cultures were used between the passage 15 and 20. THP-1 cells . The THP-1 cells were obtained from the European Collection of Authenticated Cell Cultures (THP-1 ECACC 88081201, Public Health England, UK). Cell maintenance was carried out as described in Boudish et al. [80]. All cultures were used between the passage 40 and 50. Caco-2 and LS17T cells. The Caco-2 (ECACC 86010202, Public Health England, UK) and LS174T (ECACC 87060401, Public Health England, UK) cells were obtained from the European Collection of Authenticated Cell Cultures. Caco-2 cells were maintained as described in [81]. LS174T cells were maintained in 25 cm 2 flasks with Eagle's Minimum Essential Medium (EMEM), supplemented with 1% (v/v) GlutaMax, 1% (v/v) Non Essential Amino Acids (NEAA), 10% inactivated Fetal Bovine Serum (FBS) and 1% (v/v) penicillin/streptomycin. Medium was refreshed every two days and cells were subcultured when they reached 80% confluence, as described in [81]. All cultures were used between the passage 40 and 50. Cell morphology was analyzed and checked by phase- contrast microscopy (Olympus CKX41, Olympus Corporation, Tokyo, Japan). Stimulation of HT-29 and THP-1 cell line To investigate the role of microbial shifts on NF-κB activation and innate immune response, NF-κB-HT-29-reporter cells and macrophage-like cell line (THP-1) were exposed to filtered fecal supernatant obtained from a subset of samples of each group (n=4, total n = 12) at 1 month. Samples were randomly selected among the samples with fecal material availability. Fecal supernatants prepared as described above (n=4 individuals of each group, total n = 12) were filter-sterilized (0.22 µm PES; Sarstedt SA, Barcelona, Spain) and exposed to the cells as described below. pH adjustment to 7.0-7.2 was performed when required with 0.5M filter-sterilized NaOH (Panreac, Barcelona, Spain) and buffered with HEPES (1% v/v). HT-29 cells were seeded at 7 x 10 4 cells/well in 96-wells plates and incubated for 24h in DMEM High Glucose medium without FBS supplementation. Cells were exposed to filtered fecal supernatant obtained from the three studied groups diluted in 1:10 v/v in DMEM with FBS. Supernatants were collected after 24h of stimulation and SEAP activity was measured using p-nitrophenyl phosphate according to manufacturer’s instructions (Thermo Fisher Scientist, Waltham, US). The signal was quantified using a Spectrostar Nano microplate reader (BMG Labtech, Ortenberg, Germany) at 405 nm. THP-1 cells were seeded at 5 x 10 4 in 96 wells plates in RPMI 1640, supplemented with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA). After 48 h, cells were refreshed with RPMI without PMA and incubated for 4-5 days, to allow macrophage-like differentiation of THP-1 cells. Thereafter, the cells were exposed fecal supernatants diluted 1:20 (v/v) in RPMI and incubated for 5h. Next, cell culture supernatants were collected, and cells were washed twice with PBS. Cells and supernatants were stored at -80ºC for gene expression determination and cytokine quantification, respectively. Triple co-culture in Transwell plates: Long-term stimulation A subset of samples from each group at 1 month of life (n per group=3, total n = 9) were selected to investigate in a more physiologically model the long-term effect of the differences observed in microbiota composition of the infants on the gut barrier and innate immunological state. Cell differentiation and the posterior tests were carried out in double chamber wells (Corning® Transwell®-6 well, pore size 0.4 µm; Costar, NY) equipped with separate apical and basolateral compartments and a porous support on which the Caco-2 and LS174T cells grow into a monolayer. The cells were seeded at a density of 7.5 x 10 4 cells/cm², in a proportion of 90/10 Caco-2/LS174T in supplemented DMEM. After 24 hours, apical media was refreshed, basal media was removed, and THP-1 cells were seeded in the bottom of the 6 well plate at a density of 1 ·10 5 cells/cm 2 in RPMI 1640 media containing 100 ng/mL of phorbol 12-myristate 13-acetate (PMA). After 48 hours of incubation, both apical and basolateral compartments were refreshed with DMEM or RPMI 1640 without PMA and without antibiotics, respectively. Thereafter, the triple co-culture was maintained for 4 days more and refreshments of the apical and basal media were done every 2 days. Filter-sterilized fecal supernatants selected from each studied group (n=3 individuals, total n = 9) were diluted 1:20 in cell culture media without antibiotic/antifungals and added to the apical compartment of the triple co-culture model (1.5 mL). Treatments were maintained for 7 days, with daily refreshments of the apical and basolateral compartments, with fecal supernatants or RPMI 1640, respectively. Aliquots of the culture supernatant from apical and basolateral media were stored at -80°C for cytokine measurements. Epithelial barrier function: Trans-epithelial electrical resistance (TEER) and apparent permeability. The monolayer integrity was assessed by measuring the trans-epithelial electrical resistance (TEER) and the apparent permeability (Papp) of the paracellular transport marker Lucifer yellow (LY). A Millicel-ERS (Millipore Corporation, Spain) was used for the TEER measurements. Measurements of the TEER were performed every day at the beginning of the exposure to sterilized fecal supernatant (7 days post-seeding) and every day until the end of the assay (15 days post-seeding). TEER values are reported as delta (Δ) of the initial time point before of the exposure and final time point (7 days). TEER values (ohms/cm 2 ) are presented in Additional file 9. Papp of LY was measured by adding the marker (100 µM) to the apical compartment of the wells. After 30, 60 and 120 min, 100 µL of medium was removed from the basolateral compartment and replaced with an equal volume of fresh medium (supplemented RPMI-1460 without antibiotics). LY fluorescence was measured at an excitation/emission wavelength of 485/520 nm in 96 black plates (Greiner), using a microplate fluorescence reader CLARIOstar Plus (BMG Labtech, Ortenberg, Germany). A calibration curve (0, 5, 10, 25, 50 and 100 µM) for LY quantification was run in duplicate in each reading. The Papp coefficients were calculated as previously described in [82]. Mucus production and Intestinal alkaline phosphatase (IAP) determination At day 15 post-seeding (7 days of fecal supernatant exposure), the transwell inserts containing Caco-2/LS174T cells were incubated with 1 mL of 10 mM of N-acetyl cysteine (Sigma) for 1h at 37°C, 95% humidity in DMEM, adjusted at pH 7-7.2, and the mucus produced by the cells was collected recovering the media and washing once with 0.5 mL of DMEM. The solution containing mucus was concentrated using a Vacuum Concentrator (Eppendorf, Hamburg, Germany) until dryness and re-suspended overnight in 100 µL of PBS at 4ºC. The amount of mucus was measured by a Bradford protein quantification assay, following the manufacturer instructions. Blanks containing cell culture media were subtracted and a standard curve of bovine serum albumin (BSA) was used for the calibration curve (0-5 mg/ml). Intestinal alkaline phosphatase (IAP) activity was assessed in the apical and basal compartment supernatants by enzymatic assay following manufacturer’s instructions (Sigma-Aldrich, Missouri, US) scaling the reaction to 100 µl using 4 µl of cell supernatant. Results were read in a SpectroStar Nano (BMG Labtech, Ortenberg, Germany) at 405 nm. Cytokine quantification in cell supernatant IL6, IL8 and TNF-a released by the cells after acute and long-term exposures to fecal supernatants were quantified by Enzyme-Linked ImmunoSorbent Assay (ELISA), following manufacturer instructions. Human IL8, TNF-a or IL6 Uncoated ELISA kit (Invitrogen, Carlsbad, CA, US) were used for the cytokine determination. In the long-term exposure, cytokine released values were expressed as percentage of variation comparing each treatment to control in order to avoid time-dependent effect. Samples were diluted in assay buffer to adjust the concentration to the linear range of the standard curve. Gene expression by real time RT-qPCR HT-29 and THP-1 cells from the acute exposure as well as those from tripe co-culture system were collected by scraping at the end of the treatment. RNeasy mini kit (Qiagen, Hilden, Germany) was used to RNA extraction following the manufacturer’s instructions. Total RNA was converted to cDNA by Transcriptor First Strand cDNA Synthesis Kit (Roche, Basilea Switzerland) adjusting to the same amount of RNA for each cell type. RT-qPCR analysis was performed using 1 µl of resulted cDNA reaction and 0.25 µM of the specific primers using Lightcycler 480 SYBR Green I master mix (Roche, Basilea, Switzerland). Plates were read in the LightCycler 480 (Roche, Basilea, Switzerland) at annealing temperature of 58ºC. Sequences of the primers used in the study are listed in Additional file 12 . Genes related with microbes sensing as TLRs and related transcription factors were analyzed and Actin (ACTB) gene was used as housekeeping gene expression, except for the studies in THP1 cell line in the triple co-culture system where hypoxanthine phosphoribosyl transferase (HPRT) gene was used as reference gene for showing higher stability between samples than Actin gene. Statistical analysis Chi-Squared test was used to assess differences in the categorical variables of the studied population and ANOVA or Kruskall-Wallis, followed by a Dunn’s post-hoc test, was used for the continuous variables according to the distribution of the data. Normality distribution was tested by Shapiro-Wilk test. Sequencing data were transformed to relative abundance before further analysis. Pyloseq v. 1.28.0 [83] and vegan v. 2.5-6 [84] packages were used for the analysis conducted in the sequencing data, including alpha diversity estimation. For alpha diversity analysis, samples were rarefied with a 90% of the minimum sample depth. Kruskall-Wallis followed by Dunn’s post-hoc tests were performed to detect significant differences in the gut microbial alpha-diversity according to categorical variables studies using vegan v. 2.5-6 and PMCMRplus v. 1.4.4 packages. Spearman correlations were used to find associations between alpha-diversity measures and continuous variables of the population, including gestational age or maternal body mass index, among other, and also between microbial species. For beta-diversity analysis, Permutational multivariate analysis of variance (PERMANOVA) was conducted to assess the effect of the studied factors on the neonatal gut microbial composition at ASV level (Bray-Curtis distance) and its functionality. For each factor, differences in the dispersion were also tested. In groups with different dispersions, ANOSIM test was also addressed. Calypso web platform v. 8.56 [85] was used for visualizing the multivariate analysis. The clustering of the samples according to the different studied variables, including mode and place of delivery was performed by discriminant analysis of the principal components (DAPC) at ASV level. Calypso platform was also used for Venn diagrams plotting. Kruskal Wallis test followed by a Dunn’s post-hoc test with the FDR method for multiple comparisons correction was applied to find significant differences in gut microbial composition or functionality (KEGG categories) between studied groups at each time point. For RT-qPCR analysis, LC480 Conversion version 2014.1 and LinRegPCR v. 11.0 software [86,87] were used for efficiency calculation and gene expression data were analyzed by REST2009 [88]. Statistical analysis of data from in vitro experiments and triple co-culture system was performed by Graphpad software v. 5.04 (GraphPad Software, La Jolla CA, US). Unpaired t-test was used for statistical analysis of mucus production, IAP, TEER and apparent permeability. Kruskall-Wallis and Mann-Whitney test was used for ELISA measurements, considered as non-parametric data. A p<0.05 was considered as a threshold to accept a statistically significant difference. All multiple comparisons were adjusted by false discovery rate (FDR) adjustment method. Each cell culture experiment was performed in triplicate. The R code used in this analysis are available in Additional file 13. Declarations ACKNOWLEDGEMENTS The authors thank the families involved in MAMI study as well as the contribution of the MAMI team including students, technicians and MDs who participated in obtaining the samples and preparing the metadata. We thank Teresa Gonzalo del Moral and Pepi Domínguez Cano, midwifes from Cooperativa Titània-Tascó; Marga Franch i Ferrer, midwife from Cooperativa Mudra; Concha Delgado and Adela Atero, midwifes from Casa de parts Mitjorn. Asociación de Comadronas de parto en casa de Cataluña (ALPAC), Barcelona, Spain. FUNDING This research has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC starting grant, n° 639226). M. Selma-Royo is supported by a Pre-doctoral Fellowship from Generalitat Valenciana (GVA)-European Social Fund (ASCII2016). AUTHOR CONTRIBUTORS The authors’ responsibilities were as follows: MCC: designed the study; AP, RE, CM,: recruited the families, managed the collection of the biological samples and clinical data associated; IG was coordinating the cohort information and biological samples processing and metadata; MC and MS were carried out the culture cells models and in vitro experiments. MS was responsible for the microbiota data and statistical analysis and wrote the first draft. All authors: read and approved the final manuscript. DECLARATIONS Ethics approval and consent to participate See ethics paragraph in the “Materials and methods” section. 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McMurdie PJ, Holmes S. phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data. Watson M, editor. PLoS One. 2013;8:e61217. Dixon P. VEGAN, a package of R functions for community ecology. J Veg Sci. 2003;14:927–30. Zakrzewski M, Proietti C, Ellis JJ, Hasan S, Brion M-J, Berger B, et al. Calypso: a user-friendly web-server for mining and visualizing microbiome-environment interactions. Bioinformatics. Oxford University Press; 2017;33:782–3. Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37:e45–e45. Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett. 2003;339:62–6. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. Oxford University Press; 2002;30:e36. Additional Files Additional file 1. Characteristics of studied population according to place and mode of delivery. Additional file 2. Neonatal fecal microbiota diversity and richness of meconium and infant fecal samples at 7 and 31 days. Additional file 3. Relative abundance of neonatal fecal microbiota along the first month of life Additional file 4. Taxonomic biomarkers of microbiota composition of each group depending on place and mode of delivery. Additional file 5. Core group of neonatal microbiota composition at genus level over the first moth of life. Addition file 6. Quantitative analysis of intestinal microbiota from infants born at hospital (vaginal and C-section delivery) and at home across the first month of life . Additional file 7. Microbial functions related to amino acids metabolism computationally predicted present in neonatal microbiota along the first month of life. Additional file 8. Gene Expression of HT-29 and THP-1 cells after 24 h of fecal supernatant exposure. Additional file 9. Epithelial barrier function and maturation of simulated intestinal epithelium of the triple co-culture during the long-term exposure Additional file 10. Flow chart of study participants. Additional file 11. Amplicon sequence variant (ASV) table. Additional file 12. Primers of 16s rRNA gene of prokaryotic targets and human genes tested by qPCR. Additional file 13. R code used in the study and links of consulted GitHub repositories Supplementary Files Additionalfile1.pdf Additionalfile2.pdf Additionalfile3.pdf Additionalfile4.pdf Additionalfile5.pdf Additionalfile6.pdf Additionalfile7.pdf Additionalfile8.pdf Additionalfile9.pdf Additionalfile10.pdf Additionalfile11.xlsx Additionalfile12.pdf Additionalfile13.R Cite Share Download PDF Status: Published Journal Publication published 23 Nov, 2020 Read the published version in Microbiome → Version 2 posted Editorial decision: Accept 14 Oct, 2020 Editor assigned by journal 09 Jul, 2020 Submission checks completed at journal 08 Jul, 2020 Editor invited by journal 08 Jul, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-20279","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":799106,"identity":"83fc8d39-89dc-40ec-ad8c-ce7a22779400","order_by":0,"name":"Maria Carmen Collado","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYHACxgMMDDY8UI4EcXqAWtJ4GNhI1HKYAaqFCKDbfvjAgZ8552X45zc/YPjxx0Kegb39AV4tZmfSEg72brvNI3GMzYCxt03CsIHnjAF+LTd4DA7wArUwHGMwYGZskEhgkMjB7zCQloN/t53jkT/G/oGZ4Q9Qi/xzAg4DajnMu+0Aj8ExHgNmBjaQLQwEHAb0y2HZbck8hsdyCg6C/NLGk0NAy/HDBx++3WZnL3f4+MYHP/7UyfOzH8fvMBRwAEQQHT+jYBSMglEwCnADAEOxRAS+xdbQAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6204-4864","institution":"Instituto de Agroquimica y Tecnologia de Alimentos","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Carmen","lastName":"Collado","suffix":""},{"id":799107,"identity":"ecd09f84-1fce-43ec-a4ad-61f4e4649dc9","order_by":1,"name":"Marta Selma-Royo","email":"","orcid":"","institution":"Instituto de Agroquimica y Tecnologia de Alimentos","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Selma-Royo","suffix":""},{"id":799108,"identity":"5810e2cc-61e9-4f5e-abff-078b1e06652c","order_by":2,"name":"Marta Calatayud","email":"","orcid":"","institution":"Instituto de Agroquimica y Tecnologia de Alimentos","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Calatayud","suffix":""},{"id":799109,"identity":"be4be764-b6a2-4ca7-b00e-5d789d173743","order_by":3,"name":"Izaskun García-Mantrana","email":"","orcid":"","institution":"Instituto de Agroquimica y Tecnologia de Alimentos","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Izaskun","middleName":"","lastName":"García-Mantrana","suffix":""},{"id":799110,"identity":"e0ae307b-a68d-48cc-b517-9b05773492df","order_by":4,"name":"Anna Parra-Llorca","email":"","orcid":"","institution":"Hospital Universitari i Politecnic La Fe","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Parra-Llorca","suffix":""},{"id":799111,"identity":"f05c0752-b9be-4f51-bcd7-d869c930fcbc","order_by":5,"name":"Ramón Escuriet","email":"","orcid":"","institution":"Generalitat de Catalunya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ramón","middleName":"","lastName":"Escuriet","suffix":""},{"id":799112,"identity":"8c4ca1b4-a9f3-474f-a51a-e438f62ecfa5","order_by":6,"name":"Cecilia Martínez-Costa","email":"","orcid":"","institution":"Universitat de Valencia Facultat de Medicina i Odontologia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cecilia","middleName":"","lastName":"Martínez-Costa","suffix":""}],"badges":[],"createdAt":"2020-03-30 17:28:34","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-20279/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-20279/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40168-020-00940-8","type":"published","date":"2020-11-23T15:02:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1536032,"identity":"aeed8ee5-e7df-41aa-a6d6-47f3d41069e8","added_by":"auto","created_at":"2020-07-10 20:15:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":238619,"visible":true,"origin":"","legend":"Factors affecting meconium and neonatal microbiota during the first month of life. A-C) Discriminant analysis of principal components (DAPC) of the neonatal (A) and infant fecal microbiota at 7d (B) and 31d (C) at ASV level. Each point represented microbiota from a neonate. Adonis analysis was used to stablish the significance of studied variables. D) Colonization patterns during the first moth of life. Neonatal microbiota composition at phylum level at birth (0d), 7 days (7d) and 1 month (31d). C-section (CS, n=65), vaginal delivery at hospital (VAG, n=92) and homebirth (HB, n=23).","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-20279/v2/fig1.png"},{"id":1536033,"identity":"aa650054-09f1-448f-8e1b-346c470e4ac5","added_by":"auto","created_at":"2020-07-10 20:15:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243071,"visible":true,"origin":"","legend":"Differences in relative abundance of most important and variable genera in fecal microbiota among the first month of life. Each point represented the mean and SEM of relative abundance of each genus in that point from the fecal samples of babies born by C-section (blue), vaginal delivery at hospital (green) and at home (orange). Kruskall-Wallis test with a Dunn’s post-hoc test was performed to compare the different groups. Data not sharing the same letter in each point were significantly different (p\u003c0.05). Significant variations within the same group at different time points were marked by an asterisk (*). C-section (CS), Hospitalized vaginal delivery (VAG), homebirth (HB).","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-20279/v2/fig2.png"},{"id":1536034,"identity":"0f4f18d0-69fc-4bb8-88da-6b798fc93623","added_by":"auto","created_at":"2020-07-10 20:15:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21097,"visible":true,"origin":"","legend":"Place and Mode of birth impact the infant growth. BMI z-scores (A) and Weight for Length (B) z-scores curves from delivery to 18 months of life according to mode of birth and place adjusted by covariates, breastfeeding duration, antibiotic intake during the first year of life, maternal pre-gestational BMI and infant BMI and Weight for length (W/L) z-scores at birth. General Linear Model Multivariate test adjusted by covariates was done and p\u003c0.05 was considered significant. Kruskal-Wallis was performed on the adjusted values (different letters indicate significant differences between three studied groups). C-section (CS, n=58), Hospitalized vaginal delivery (VAG, n=85), homebirth (HB, n=23). ","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-20279/v2/fig3.png"},{"id":1536035,"identity":"dcdb07f8-6a39-46ca-80fa-62c025aecb20","added_by":"auto","created_at":"2020-07-10 20:15:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":244461,"visible":true,"origin":"","legend":"Microbial functions computationally predicted present in neonatal microbiota along the first month of life. A-B) Discriminant analysis of principal components (DAPC) of the neonatal (A) and infant fecal microbiota at 7d and 31d (B) Adonis analysis was used to stablish the significance of studied variables. C-D) Computational analysis of lipopolysaccharide (LPS) biosynthesis (C) bacterial toxins (D) routs presents in the fecal microbiota of newborns along the first month of life. Results were expressed as percentage of total functional routs for each sample. *p\u003c0.05, **p\u003c0.01, ***p\u003c0.001. C-section (CS, n=65), Hospitalized vaginal delivery (VAG, n=92), homebirth (HB, n=23). ","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-20279/v2/fig4.png"},{"id":1536036,"identity":"acebb8c2-32a9-407b-ad74-3386509d92e0","added_by":"auto","created_at":"2020-07-10 20:15:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26018,"visible":true,"origin":"","legend":"Effect of 1-month infant fecal water exposure in epithelial (A) and macrophages-like (B) cell lines after 24h. A) Cytokine production by HT-29 cells after exposure to fecal water from neonates born by C-Section (CS), vaginal delivery at hospital (VAG) and Homebirth (HB). IL6 production in HT-29 cell line was below detection limit. B) Cytokine production of THP1 cells after 24h exposure to fecal supernatants of each group. Data was presented as median and whiskers represented the 5-95 percentile. Kruskal-Wallis and Dunn’s post hoc (FDR adjustment) test was used to test the significance of the differences in cytokine response between the groups. *p\u003c0.05, **p\u003c0.01, ***p\u003c0.001. C-section (CS), Hospitalized vaginal delivery (VAG), homebirth (HB).","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-20279/v2/fig5.png"},{"id":1536037,"identity":"001c01a0-8226-4eef-b785-afde4a23c9bc","added_by":"auto","created_at":"2020-07-10 20:15:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":188632,"visible":true,"origin":"","legend":"Effect of fecal water long-term exposure (7d) on the triple co-culture system. A-B) Epithelial barrier function measured as trans-epithelial electric resistance (TEER) (A) and Lucifer Yellow transport (LY) (B). C) Mucus production by LSTH17 cells after the long-term exposure on the triple co-culture system measured by Bradford assay. D) Interleukin (IL) 8 production by cells on the apical compartment (Caco-2 and LSTH17) measured by ELISA and expressed as increment respect to control condition. E-F) Intestinal cells functional maturation degree measured as intestinal alkaline phosphatase activity (IAP) on apical compartment during the treatment (E) and at final time point (F). G-H) Cytokine production in the basal compartment by THP-1 cells. IL- 8 (G) and IL-6 (H) production after fecal supernatant long-term exposure expressed as increment respect to control condition. \nThe treatments were fecal water from infants born by C-section (CS), vaginal delivery at hospital (VAG) and homebirth (HB). Non-normal data was presented as median and whiskers represented the 5-95 percentile while normal data was showed as mean and SD. Kruskal-Wallis/Anova and Dunn’s/Tukey’s post hoc (FDR adjustment) test was used to test the significance of the normal/non normal distributed variables between the groups. In the cytokine analysis, the symbol (*) represented variations between time within the same studied group according to the color. *p\u003c0.05, **p\u003c0.01, ***p\u003c0.001.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-20279/v2/fig6.png"},{"id":13550509,"identity":"72f7b597-43ca-4c8c-af0b-01fc845d5863","added_by":"auto","created_at":"2021-09-17 02:25:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1687224,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-20279/v2/68b900cf-0807-4475-b9fb-2d82cc38a323.pdf"},{"id":1536039,"identity":"c917d09f-f3cb-4eda-be55-b9adb336b61f","added_by":"auto","created_at":"2020-07-10 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20:15:20","extension":"r","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":5579,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile13.R","url":"https://assets-eu.researchsquare.com/files/rs-20279/v2/Additionalfile13.R"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePerinatal Environment Shapes Microbiota Colonization And Infant Growth: Impact On Host Response And Intestinal Function \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobial colonization plays an important role in numerous functions, including digestion, metabolic reactions and trophic effects, and it also influences the development and maturation of the host\u0026rsquo;s innate and adaptive immune system [1\u0026ndash;3]. The mode of birth is a key factor shaping early microbial colonization [4\u0026ndash;6]. Vaginally born (VAG) infants acquire microbial communities resembling the maternal vaginal and gut microbiota, whereas infants born via C-section acquire environmental-like bacteria such as \u003cem\u003eStaphylococcus, Corynebacterium\u003c/em\u003e and \u003cem\u003ePropionibacterium\u003c/em\u003e spp. [7]. In addition, CSs are associated with lower microbial diversity, delayed colonization of \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e spp. and reduced immune responses [8]. The CS rate increased by an annual increment of 3.7% from 2000 to 2015 worldwide \u0026nbsp;[9,10]. In Europe, the average CS rate is 28% [11], although the World Health Organization (WHO) recommends a rate of 10\u0026ndash;15% [12]. Epidemiological studies have linked CSs with a higher risk of non-communicable diseases such as obesity [13,14] and allergy [15]. Indeed, the CS procedure is characterized by pre- and intra-partum antibiotic exposure and other medical practices, which may affect early gut colonization and predispose the infant to developing immune-related disorders later in life, including asthma [16\u0026ndash;18], allergy [19\u0026ndash;21], obesity [22,23] and diabetes [24,25]. Hospital interventions during birth are critical for pioneer microbial colonizers and proper immune system maturation [26,27], which may impact adult health [28,29]. However, neonatal microbiota colonization in the absence of hospital interventions remains underexplored. Furthermore, the hospital environment (high-level disinfection and antibiotic therapy) has an impact on microbial exposure, thereby extending the hygiene hypothesis to the time of birth [30,31].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHome births (HBs) increased by 77% between 2004 and 2017 in the United States, while the rate of birth-center deliveries doubled during the same period [32]; however, in Europe, such births account for less than 1% of all deliveries, except in the Netherlands, where HBs represent 16.3% of births [33]. Recently, a distinct microbiota profile has been reported in VAG neonates born at home or in hospital [34], although the impact on microbiota development and the potential effects on neonatal health are not fully understood.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigate the influence of birth-related factors, including the place and mode of delivery on early colonization during the first month of life and on infant growth during the first 18 months. Furthermore, to understand the potential biological mechanisms involved, \u003cem\u003ein vitro\u003c/em\u003e gut models are used to study the impact of distinct microbiota patterns on intestinal function and innate immune system maturation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStudy population \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo differences were observed in the neonatal weight between groups, which showed a median of 3250 g (range 2973\u0026ndash;3573 g). Other maternal clinical parameters are presented in the supplementary material (Additional file 1). Despite all the babies were born at full term, the CS-born neonates were born before the both groups of vaginally delivered neonates (39 weeks of pregnancy for CS births and 40 weeks for the VAG and HB neonates, respectively) (\u003cem\u003ep=0.004\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eThe HB neonates showed higher length measurements than the hospital-born infants for both delivery modes (\u003cem\u003ep=0.003\u003c/em\u003e). Additionally, the HB infants had higher ratios of exclusive breastfeeding than the hospital-delivered infants at both seven and 31 days of life (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerinatal factors related to the place and mode of delivery shape the neonatal microbiome composition at birth\u0026nbsp; \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt birth, the place (hospital versus home) was the main contributor of neonatal microbiota composition (\u003cem\u003ep=0.001\u003c/em\u003e), followed by the mode of birth (\u003cem\u003ep=0.025\u003c/em\u003e) (Figure, 1A). Other perinatal factors did not significantly influence the neonatal microbiota during delivery.\u003c/p\u003e\n\u003cp\u003eThe place and mode of birth shaped the microbial richness and diversity at amplicon sequence variant (ASV) level (Additional file 2). The hospital-born neonates showed a bacterial community with greater richness (\u003cem\u003ep=0.002\u003c/em\u003e) and diversity (\u003cem\u003ep=0.072\u003c/em\u003e) at birth than the HB neonates. The CS-born neonates harbored higher index of observed species (\u003cem\u003ep=0.023\u003c/em\u003e), diversity (\u003cem\u003ep=0.001\u003c/em\u003e) and richness (\u003cem\u003ep=0.031\u003c/em\u003e) than the HB infants.\u003c/p\u003e\n\u003cp\u003eThe neonatal fecal microbiota was dominated by the Proteobacteria phylum, followed by Firmicutes and Actinobacteria (Additional file 3). The hospital-born infants showed higher relative abundances of the \u003cem\u003eFinegoldia\u003c/em\u003e (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e), \u003cem\u003eClostridioides\u003c/em\u003e (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e), \u003cem\u003eKlebsiella\u003c/em\u003e (\u003cem\u003ep=0.025\u003c/em\u003e) and \u003cem\u003ePeptoniphilus\u003c/em\u003e (\u003cem\u003ep=0.006\u003c/em\u003e) genera, including \u003cem\u003eClostridioides difficile\u003c/em\u003e (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e) and \u003cem\u003eClostridium neonatale \u003c/em\u003e(\u003cem\u003ep=0.006\u003c/em\u003e), while the HB infants showed higher relative abundances of the \u003cem\u003eStaphylococcus\u003c/em\u003e (\u003cem\u003ep\u0026lt;0.007\u003c/em\u003e) and \u003cem\u003eEnterococcus\u003c/em\u003e (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e) genera.\u003c/p\u003e\n\u003cp\u003eAs for the mode of birth, the VAG infants showed higher relative abundances of the Bacteroidetes (\u003cem\u003ep=0.035\u003c/em\u003e) and Firmicutes (\u003cem\u003ep=0.035\u003c/em\u003e) phyla, including the \u003cem\u003eBacteroides \u003c/em\u003eand\u003cem\u003e Escherichia/Shigella\u003c/em\u003e genera. A linear discriminant analysis of effect size (LEfSe) was used to confirm which genera were responsible for the clustering of the rectal swab microbial population (Additional file 4). The microbiota of the HB neonates was enriched by species from the \u003cem\u003eEnterococcus \u003c/em\u003eand \u003cem\u003eStaphylococcus \u003c/em\u003egenera, while the hospital-born infants were enriched with species from the \u003cem\u003eKlebsiella\u003c/em\u003e, \u003cem\u003eVeillonella\u003c/em\u003e and \u003cem\u003eClostridioides\u003c/em\u003e genus. We also observed a microbial core at each time point and noted some unique genera not shared between the groups (Additional file 5). For instance, we observed that the \u003cem\u003eAkkermansia \u003c/em\u003egenus was only present in the HB.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerinatal factors related to the place and mode of delivery shape neonatal microbiota development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMode of birth (\u003cem\u003ep=0.001\u003c/em\u003e) and time (\u003cem\u003ep=0.001\u003c/em\u003e) were the main contributors to the overall microbiota structure during the first month of life at ASV level. Generally, neonatal microbiota at birth showed significantly higher microbial richness (Chao1 index) and diversity (Shannon index) than the microbiota at 7 and 31 days of life (Additional file 2). No differences in alpha diversity (Chao 1, Shannon index) were found according to the place or mode of delivery at the 7- or 31-day time points.\u003c/p\u003e\n\u003cp\u003eDistinct colonization patterns were identified between the hospital born (VAG and CS) and HB neonates (Figure, 1D). The mode of birth shaped the microbiota colonization process at 7 (Adonis\u003cem\u003e p\u0026lt;0.001\u003c/em\u003e) and 31 days (Adonis\u003cem\u003e p\u0026lt;0.005\u003c/em\u003e) at ASV level.\u003c/p\u003e\n\u003cp\u003eThe infant microbiota showed differing development in the VAG and CS-delivered neonates at the phylum and family levels (Figure, 1D). The VAG deliveries exhibited a colonization pattern somewhere between the patterns of the CS-born and HB deliveries. The HB and CS-born infants showed significant differences in the Firmicutes phylum at delivery (\u003cem\u003ep=0.035\u003c/em\u003e) and in the Proteobacteria at seven days (\u003cem\u003ep=0.019\u003c/em\u003e), although they did not show differences when compared to the hospital-based VAG infants. The relative abundance of the Actinobacteria phylum increased during neonatal life in the both groups of vaginally (VAG and HB) born infants but not in the CS-born neonates (Additional file 3). In the HB neonates, the Actinobacteria increase occurred from delivery to 31 days, while in the hospital-based VAG neonates, the increase was delayed from seven to 31 days of life. The Bacteroidetes phylum abundance was slightly higher in the vaginal births (VAG and HB) at seven (\u003cem\u003ep=0.014\u003c/em\u003e) and 31 days (\u003cem\u003ep=0.01\u003c/em\u003e) than in the CS births.\u003c/p\u003e\n\u003cp\u003eAt the phylum level, the vaginal births (VAG and HB) showed higher relative abundances of the Actinobacteria (\u003cem\u003ep=0.001\u003c/em\u003e) and Bacteroidetes (\u003cem\u003ep=0.018\u003c/em\u003e) phyla, especially \u003cem\u003eBifidobacterium\u003c/em\u003e (p=0.003 at seven days), when compared with the CS-born infants at both times (7and 31 days). The CS-born infants were enriched with the Firmicutes phylum (\u003cem\u003ep=0.020\u003c/em\u003e), including \u003cem\u003eEnterococcus\u003c/em\u003e (\u003cem\u003ep=0.005 \u003c/em\u003eat seven days) and \u003cem\u003eClostridium \u003c/em\u003e(\u003cem\u003ep=0.039\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eThe HB neonates harbored a higher relative abundance of Actinobacteria (\u003cem\u003ep=0.004 \u003c/em\u003eand \u003cem\u003ep=0.006\u003c/em\u003e at 7 and 31 days, respectively) and a lower relative abundance of Proteobacteria (\u003cem\u003ep=0.026 \u003c/em\u003eand \u003cem\u003ep=0.008\u003c/em\u003e at 7 and 31 days, respectively) when compared with the hospital-born infants (VAG and CS) at both times. Hospital-born infants had higher relative abundances of the \u003cem\u003eKlebsiella\u003c/em\u003e species and lower relative abundances of \u003cem\u003eBifidobacterium \u003c/em\u003egenus, including \u003cem\u003eB. bifidum\u003c/em\u003e (\u003cem\u003ep=0.013\u003c/em\u003e), and \u003cem\u003eCollinsella\u003c/em\u003e genus, including \u003cem\u003eCollinsella aerofaciens\u003c/em\u003e (\u003cem\u003ep=0.028\u003c/em\u003e at 31 days), compared with the HB neonates.\u003c/p\u003e\n\u003cp\u003eIn the vaginal births (both, VAG and HB), the relative abundances of the \u003cem\u003eCollinsella\u003c/em\u003e and \u003cem\u003eBacteroides \u003c/em\u003egenera increased during the first month of life. An opposite trend was observed for the \u003cem\u003eEscherichia\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e genera (Figure 2). In the CS-born infants, \u003cem\u003eEscherichia\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e genera increased from birth to seven and 31 days (Figure 2). The relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e genus was higher in the vaginal births, especially the HB infants at 7 (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e) and 31 days (\u003cem\u003ep=0.004\u003c/em\u003e), when compared with the CS births.\u0026nbsp; Moreover, CS-neonates harbored a higher relative abundance of the \u003cem\u003eClostridium sensu stricto\u003c/em\u003e genus than the HB infants at 7 days (\u003cem\u003ep=0.007\u003c/em\u003e), although no difference was observed with the hospital-based VAG infants (\u003cem\u003ep=0.250\u003c/em\u003e), indicating intermediate colonization patterns in neonates born in hospital via vaginal route.\u003c/p\u003e\n\u003cp\u003eAt both 7 and 31 days, the \u003cem\u003eBacteroides \u003c/em\u003egenus was present between the vaginal-delivered neonates but not the CS-delivered infants, who showed \u003cem\u003eEnterobacter\u003c/em\u003e as a unique genus at 31 days.\u003c/p\u003e\n\u003cp\u003eIn a subset of samples, total bacterial counts by qPCR were significantly lower at birth than the counts obtained at seven days in all three groups (Additional file 6). We also found that the CS-born fecal samples showed significantly lower bacterial counts than the VAG (\u003cem\u003ep=0.043\u003c/em\u003e) and HB (\u003cem\u003ep=0.008\u003c/em\u003e) samples at delivery. The HB infants showed higher total bacterial counts than the VAG (\u003cem\u003ep=0.008\u003c/em\u003e) and CS-born (\u003cem\u003ep=0.043\u003c/em\u003e) infants at seven days. Similarly, in the case of the \u003cem\u003eBifidobacterium\u003c/em\u003e genus counts in the vaginal-delivered groups (HB [\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e] and VAG [\u003cem\u003ep=0.003\u003c/em\u003e]), the counts increased during the first month. Furthermore, the HB infants had higher \u003cem\u003eBifidobacterium\u003c/em\u003e counts than the VAG and CS-born infants at seven (\u003cem\u003ep=\u0026lt;0.001\u003c/em\u003e for CS, \u003cem\u003ep=0.004\u003c/em\u003e for VAG) and 31 days (\u003cem\u003ep=0.027\u003c/em\u003e for CS, \u003cem\u003ep=\u0026lt;0.001\u003c/em\u003e for VAG).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of the perinatal environment on the infant weight status at 18 months \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigher BMI and W/L z-scores were observed in the CS-born infants than in the HB (\u003cem\u003ep=\u0026lt;\u003c/em\u003e0.001 for BMI, \u003cem\u003ep\u0026lt;\u003c/em\u003e0.001 for W/L at 12 months) and hospital-born vaginal births (\u003cem\u003ep=\u0026lt;\u003c/em\u003e0.001 for BMI and \u003cem\u003ep=\u003c/em\u003e0.003 for W/L at 12 months) (Figure, 3 A-B). Indeed, at 18 months of life, CS infants exhibit also higher BMI z-scores than HB (\u003cem\u003ep\u0026lt;\u003c/em\u003e0.001) and VAG \u003cem\u003e(p=\u003c/em\u003e0.016) children. Additionally, a multivariate linear analysis (adjusted by breastfeeding duration, antibiotic intake during the first year of life, maternal pre-gestational BMI, and BMI and W/L z-scores at delivery) showed the CS-born neonates to exhibit significantly higher BMI and W/L z-scores across the first 18 months of life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobiota predicted functionality\u003c/strong\u003e\u003cstrong\u003e during the first month of life is influenced by the birth mode and place \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inferred microbial functionality at birth was mainly affected by the mode of birth (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e), but also by the birthplace (\u003cem\u003ep=0.049\u003c/em\u003e). In addition, mode of birth also influenced the microbiota predicted functionality at 7 (\u003cem\u003ep=0.001\u003c/em\u003e) and 31 days (\u003cem\u003ep=0.02\u003c/em\u003e); however, the birthplace was only significant at 7 days (\u003cem\u003ep=0.001\u003c/em\u003e) (Figure 4, A-B).\u003c/p\u003e\n\u003cp\u003eRegarding concrete functions, the microbiota of the vaginal-delivered infants (VAG and HB) were enriched in functional routes related to the synthesis of secondary metabolites (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e), amino acids (\u003cem\u003ep=0.002\u003c/em\u003e), lipids (\u003cem\u003ep=0.034\u003c/em\u003e) and carbohydrate metabolism (\u003cem\u003ep=0.046\u003c/em\u003e) when compared with the CS-born neonates at delivery (Additional file 7). At seven and 31 days, the vaginal-delivered infants showed microbiota enriched in lysine, phenylalanine, tyrosine, tryptophan, valine, leucine and isoleucine biosynthesis pathways. At one month of life, the CS-born infants expressed higher energy metabolism pathways, including carbohydrates, lipids, and propanoate biosynthesis.\u003c/p\u003e\n\u003cp\u003eThe immune-system-related paths (e.g. antigen processing and presentation [\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e] and NOD-like receptors [\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e]) were overrepresented in the CS-born infants at birth. Contrarily, the lipopolysaccharide (LPS) biosynthesis routes were enriched in the vaginal-delivered infants at all time points (Figure 4, C-D), being also influenced by the birthplace. The genera that mostly contribute to the differences in these predicted routs were \u003cem\u003eEscherichia/Shigella\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e and \u003cem\u003eClostridium sensu stricto\u003c/em\u003e. However, the CS-delivered infants showed increasing representation of pathways related to bacterial toxins at seven (\u003cem\u003ep=0.011\u003c/em\u003e) and 31 days (\u003cem\u003ep=0.004\u003c/em\u003e) when compared with the HB neonates.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFecal supernatants induce the mRNA expression of \u003cem\u003eTLR4\u003c/em\u003e and \u003cem\u003eIRAK4\u003c/em\u003e in intestinal epithelial cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HT-29 reporter cells showed increased NF-kB activation independently of the place and mode of delivery in all the groups (data not shown). All fecal supernatants induced IL8 and TNF-a production in the HT-29 cells when compared with control cells exposed to cell culture media (Figure 5, A). No differences were found between the groups in relation to any of the cytokines.\u003c/p\u003e\n\u003cp\u003eHB fecal supernatants up-regulated the \u003cem\u003eTLR4\u003c/em\u003e mRNA in a 1.405-fold change (\u003cem\u003ep=0.024\u003c/em\u003e) and the \u003cem\u003eIRAK4\u003c/em\u003e mRNA in a 2.654-fold change (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e) when compared with the control samples. This increase was not observed in the cells exposed to hospital-born (VAG and CS) neonatal samples (Additional file 8). Generally, the fecal supernatants all reduced the mRNA expression of the tight-junction proteins, including zonulin-1 (\u003cem\u003eHP\u003c/em\u003e) (0.202- and 0.265-fold change in the HB and CS samples, respectively), e-cadherin (\u003cem\u003eCDH1\u003c/em\u003e) (0.226- and 0.326-fold change in the HB and CS, respectively) and occludin (\u003cem\u003eOCLN\u003c/em\u003e) (0.192- and 0.253-fold change in the HB and CS). However, no differences were found between the different studied groups.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHB fecal supernatants trigger higher immune response in macrophage-like cells than CS fecal supernatant. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mode of birth significantly affected the production of cytokines in the PMA-differentiated THP-1 cells (Figure 5, B). The HB and VAG fecal supernatants had a higher pro-inflammatory capacity than the CS samples. Higher levels of IL6 and IL8 were detected after HB (\u003cem\u003ep=\u0026lt;0.001\u003c/em\u003e), followed by the VAG (\u003cem\u003ep=0.043\u003c/em\u003e) samples, when compared with the CS-born fecal supernatant. The VAG and HB samples triggered a significantly higher response than the control condition for IL6, IL8 and TNF-a, which was not observed in cells exposed to CS samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe CS fecal supernatants down-regulated the \u003cem\u003eTLR4 \u003c/em\u003e(0.509-fold change, \u003cem\u003ep=0.006\u003c/em\u003e) and \u003cem\u003eFOS\u003c/em\u003e mRNA expression (0.238-fold change, \u003cem\u003ep=0.038\u003c/em\u003e) (Additional file 8). The expression levels were not quantifiable for the interferon gamma (\u003cem\u003eIFN-\u003c/em\u003e\u003cem\u003eg\u003c/em\u003e) and \u003cem\u003eIL10\u003c/em\u003e genes in either the HT-29 or THP-1 cell lines following faecal supernatant exposure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTriple co-culture system for host\u0026ndash;microbiome interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePlace and mode of birth impacts on intestinal barrier function and maturation \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWith regard to the integrity of the cell monolayer (Additional file 9), HB promoted a higher increase in the transepithelial electrical resistance (TEER) values after seven days of exposure (Figure 6, A-B) when compared with hospital birth (VAG [\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e] and CS [\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e]). These results were confirmed via the measurement of lucifer yellow dye (LY) transport through the epithelial layer. Despite all the fecal supernatants triggered an increase in LY transport when compared to control condition (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e), hospital-based birth, both VAG (2.1\u0026middot;10\u003csup\u003e-6\u003c/sup\u003e\u0026plusmn;5.7\u0026middot;10\u003csup\u003e-7 \u003c/sup\u003ecm/s, \u003cem\u003ep=0.018\u003c/em\u003e) and CS (2\u0026middot;10\u003csup\u003e-6\u003c/sup\u003e\u0026plusmn;3.5\u0026middot;10\u003csup\u003e-7\u003c/sup\u003e cm/s, \u003cem\u003ep=0.033\u003c/em\u003e), led to higher LY transport than HB (1.5\u0026middot;10\u003csup\u003e-6\u003c/sup\u003e\u0026plusmn;1.7\u0026middot;10\u003csup\u003e-7\u003c/sup\u003e cm/s).\u003c/p\u003e\n\u003cp\u003eThe intestinal alkaline phosphatase (IAP) activity was also measured in the supernatant of cell cultures from both; the apical and basal compartments (Figure 6, D). Indicative of functional cell polarization, the IAP activity was significantly higher (\u003cem\u003ep=0.006\u003c/em\u003e) in the apical compartment at 7 days of treatment, while all the fecal supernatants enhanced the IAP activity when compared with the control. Similar to the TEER and LY results, HB samples induced higher IAP activity than the ones observed in VAG (\u003cem\u003ep=0.043\u003c/em\u003e) and CS samples (\u003cem\u003ep=0.049\u003c/em\u003e). Furthermore, higher mucus production was observed in cells exposed to hospital fecal samples (0.92\u0026plusmn;0.2 mg/ml) when compared with the HB samples (0.69\u0026plusmn;0.09 mg/ml) (\u003cem\u003ep=0.006\u003c/em\u003e).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmune system response\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe immune response to the fecal supernatants was generally higher in the basal than in the apical compartment. In the simulated intestinal epithelium, the exposure to CS-born fecal supernatants down-regulated (0.43-fold change) the \u003cem\u003eIRAK4\u003c/em\u003e mRNA expression when compared with the control-no stimulus (\u003cem\u003ep=0.01\u003c/em\u003e) and HB samples (0.45-fold change, \u003cem\u003ep=0.001\u003c/em\u003e). Contrarily, the CS-born fecal supernatants up-regulated the toll-interacting protein (\u003cem\u003eTOLLIP)\u003c/em\u003e mRNA (\u003cem\u003ep=0.007\u003c/em\u003e) expression in the HB samples (1.54-fold change) (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:-49.7pt;margin-bottom:.0001pt;margin-left:0in;font-size:12px;font-family:\"Calibri\",sans-serif;color:#1F497D;font-style:italic;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 13px; font-family: Helvetica; color: rgb(0, 0, 0); font-style: normal;\"\u003eTable\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-style: normal;\"\u003e1\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-style: normal;\"\u003e. Gene expression of cells in the apical and basal compartment in the triple co-culture system after 7 days of exposure\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-style: normal;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"float: left;width:474.9pt;border-collapse:collapse;border:none;margin-left:4.8pt;margin-right:4.8pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 159.5pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; HB\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 235pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; CS\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 20.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 20.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003eFold expression\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 20.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 20.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003eFold expression\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 20.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u0026nbsp; p-value\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" style=\"width: 474.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.7pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eApical compartment (Caco-2, LSTH-17 cells)\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;border: none;background: silver;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eHP\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;border: none;background: silver;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.139 (0.767 - 1.627)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;border: none;background: silver;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.495\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;border: none;background: silver;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.097 (0.957- 1.214)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: none;background: silver;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.106\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eCDH1\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.054 (0.462 - 1.675)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.841\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e0.916 (0.736-1.237)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.399\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eOCLN\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.163 (0.673 - 1.534)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.531\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.321 (0.867-1.702)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.083\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eIRAK4 (#)\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e0.94 (0.610 - 1.168)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.837\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e0.425 (0.273-0.595)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003e0.010* \u0026darr;\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eTLR2\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.492 (0.837 - 2.142)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.181\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.302 (0.960-1.710)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.061\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eTLR4\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e0.89 (0.722 - 1.074)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.201\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e0.826 (0.482-1.276)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;padding: 0in 5.4pt;height: 13.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.475\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eTOLLIP\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e0 (0.000 - 1.381)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.509\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.231 (0.658-2.076)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.474\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 411.1pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eBasal compartment (THP-1 cells)\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;border: none;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eTLR4\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;border: none;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e3.566 (1.103 - 13.629)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;border: none;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.094\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;border: none;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.117 (0.235 - 7.013)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.896\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eTLR3\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.346 (0.939 - 1.853)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.378\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e3.546 (1.712 - 8.099)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cem\u003e0.094\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eTOLLIP\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e5.182 (3.667 - 9.351)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0 .001*\u0026uarr;\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e7.186 (4.388 - 11.727)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: silver;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003e0.010*\u0026uarr;\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003eIL10\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e7.888 (3.496 - 31.769)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 70.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt; 0.001*\u0026uarr;\u003c/em\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style=\"font-size: 13px;\"\u003e\u003cspan style=\"font-family: Helvetica;\"\u003e1.737 (0.912 - 5.478)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height: normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Helvetica; font-size: 13px; color: rgb(0, 0, 0);\"\u003e\u003cem\u003e0.061\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cp style='margin-top:0in;margin-right:-49.7pt;margin-bottom:.0001pt;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style=\"font-size: 13px; line-height: 115%; font-family: Helvetica; color: rgb(0, 0, 0);\"\u003eTotal RNA was extracted from cells treated with homebirth (HB) samples and C-section (CS) fecal supernatant. For each condition, n=6 in triplicates, data was presented as fold change expression (95% C.I). \u0026nbsp;Values expressed relative expression fold-change of each condition compared to control. \u003cem\u003eP\u0026lt;0.05\u003c/em\u003e (*) and blond letters marked significant differences between treatment and control, up (\u0026uarr;) or down (\u0026darr;) regulation was represented by the arrows. Symbol # represented genes expression that was different between HB and CS. \u003c/span\u003e\u003c/p\u003e\u003cbr\u003e\n\u003cp\u003eIL8 was detectable at all time points in the apical compartment (Figure 6, E). Generally, the IL8 exhibited a gradual increase until the fifth day of exposure (126\u0026plusmn;39% average increase for the three groups). Thereafter, the IL8 levels decreased until the end of treatment (7 days, 99\u0026plusmn;24% average of three groups). The IL6 concentration was only above the detection limit at the final time point (7days), and there were no differences between the groups.\u003c/p\u003e\n\u003cp\u003eIn the basal compartment, similar to the epithelium-like layer, we observed a 7.2-fold change in the down-regulation of the \u003cem\u003eTOLLIP\u003c/em\u003e mRNA expression after exposure to CS-born samples (\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e) (Table 1).\u003c/p\u003e\n\u003cp\u003eIL8 release was observed at 24 hours (151.3\u0026plusmn;42.7% increase from the control), with the maximum being seen after five days of treatment (164\u0026plusmn;65% increase). At day seven, the IL8 levels reached similar values to those seen on the first day of treatment (82\u0026plusmn;67% increase) (Figure 6, F-J).\u003c/p\u003e\n\u003cp\u003eThe IL6 production by the THP-1 cells showed a late response to faecal supernatant exposure, lasting from day one of treatment until day three (218\u0026plusmn;223% increase from control); however, the increase was only significant for the HB (\u003cem\u003ep=0.003\u003c/em\u003e) and VAG (\u003cem\u003ep=0.017\u003c/em\u003e) samples. At the subsequent time points, the IL6 release was stabilised to concentrations similar to those seen on day one (61.45\u0026plusmn;11.69% for CS, 48.88\u0026plusmn;11.14% and 59.97\u0026plusmn;13.4% for HB). No significant differences were observed between the groups in terms of the cytokine production patterns over the seven days.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated gut microbiota evolution during the first month of life in CS-delivered and both HB and hospital-based VAG infants. Our results highlight the relevance of perinatal factors to the gut microbial colonization pattern, which affects the innate defensive mechanisms and functional maturation at the intestinal level.\u003c/p\u003e\n\u003cp\u003eCS procedures are associated with specific conditions, including the use of antibiotics, longer hospitalization, low neonatal\u0026ndash;maternal contact and the delayed initiation of breastfeeding [35,36], which may influence microbial colonization patterns [26]. A higher percentage of bottle or mixed feeding is commonly observed in CS-born infants [35], including the studied cohort. In our cohort, the HB infants showed a higher rate of exclusive breastfeeding than the hospital-born infants (95% in HB versus 67% and 61% in CS and VAG at hospital, respectively). Furthermore, the practices associated with hospital delivery, as antibiotic use, vaginal cleansing, controlled maternal food intake and mobility, oxytocin administration or anesthesia, may affect the maternal microbiota and, consequently, mother\u0026ndash;infant microbial transmission. Those factors together with prematurity would have a relevant impact on infant development that remains poorly understood. Additionally, these birth-related factors may affect the microbial establishment process and cannot be independently studied. Therefore, our results sum up the consequences of all the concomitant factors affecting the different groups of study.\u003c/p\u003e\n\u003cp\u003eThe hospital environment, especially in the case of CSs, can be considered highly intervened condition characterized by the high pressure of antibacterial therapy and instrumentalization. HB rates have increased in Europe over recent decades, ranging from 0.1% in Sweden to almost 20% in the Netherlands [37]. However, limited information concerning the possible benefits of HB is currently available due to the lack of adequate randomized clinical trials meaning that it is not possible to determine the risk of neonatal\u0026ndash;maternal mortality and morbidity during HB [38]. Further, little is known about the impact of a non-hospital environment on infant gut colonization.\u003c/p\u003e\n\u003cp\u003eWe observed that the mode and place (hospital versus home) of birth shaped the neonatal microbiota. The microbiota of hospital-delivered infants was enriched with gram-positive anaerobic cocci, including the \u003cem\u003ePeptoniphilus\u003c/em\u003e and \u003cem\u003eFinegoldia\u003c/em\u003e genera, while those of HB infants exhibit higher relative abundances of species from the \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e genera.\u003c/p\u003e\n\u003cp\u003eIn all three groups, time was the main factor affecting the composition of the infant microbiota, with a significantly different pattern between groups observed at birth, but not at the subsequent time points. Primary events concerning gut microbial colonisation may impact the assembly and acquisition of the early microbiome, likely having long-lasting consequences for gut ecology [29].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt delivery, we observed higher diversity indices in the neonatal microbiota than at the subsequent time points; however, the quantitative total bacteria count was lower at delivery. This is likely caused by bias in the sequencing data obtained from low biomass samples and by higher contact with environmental contaminants. The HB infants had lower microbial diversity but a higher total number of bacteria than the hospital-born infants, likely due to the latter having increased contact with bacterial environmental contaminants [6].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe found clearly identifiable differences in the global microbiota structure during time between the three groups, even at the phylum level, which is in agreement with previous studies [4,6,34]. Interestingly, the differences in the colonization patterns become more pronounced over time, at least during the first month of life, indicating the importance of birth-related events to neonatal colonization processes, which may affect the microbiome composition in later developmental stages. Altered microbial colonization has been also observed in preterm infants compared to term infants, and these microbial shifts have been mainly linked to antibiotics exposure, caesarean section, hospitalization, use of formula feeding. Regarding term infants, some studies have shown shifts related to delivery mode in term infant microbiota up to 3 months post-delivery [8,39], other authors observed no differences in terms of microbiota composition in neonates born by CS beyond the first days of life [40]. Thus, the duration of the shifts in infant microbiota associated to delivery mode has reported contradictory results and needs to be further evaluated.\u003c/p\u003e\n\u003cp\u003eSimilar to prior studies, we found that CS-born infants showed higher relative abundances of \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e and lower species from the\u003cem\u003e Bifidobacterium\u003c/em\u003e genus [6]. We observed that both VAG and HB infants had higher number of \u003cem\u003eBifidobacterium\u003c/em\u003e species than CS-born neonates. The depletion of the \u003cem\u003eBifidobacterium\u003c/em\u003e species in the gut environment has been associated to immune-related diseases [41,42], and members of this genus are commonly used as probiotics due to their capacity to modulate microbiota\u0026ndash;immune system homeostasis [43].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCould these shifts in the microbiota composition alter the functional profile of the neonatal microbiota? \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDescriptive and observational studies are relevant to understanding microbiome evolution during the neonatal period; however, mechanistic studies of the host\u0026ndash;microbiome interplay during early life are still required. Thus, microbiota functional analysis has been proposed as a tool for clarifying the host\u0026ndash;microbiome interactions [44]. Our results of the predicted metagenome from 16S rRNA sequencing data showed functional differences between the place and mode of birth in the infant microbiota at delivery, 7 and 31 days. Several amino acid (AA) biosynthesis routes were over-represented in the microbiota from vaginal-delivered infants when compared with the CS-born infants, including tryptophan-related paths. It is known that AAs serve as regulators of several metabolic pathways in the host [45] and more specifically, tryptophan interacts with the immune system through microbial serotonin production [46], regulation of TLRs or interacting with the aryl hydrocarbon receptor\u0026ndash;microbiota\u0026ndash;immune system path [47,48].\u003c/p\u003e\n\u003cp\u003eWe also found that vaginal-delivered infants, especially HB babies, had a microbiota enriched with LPS biosynthesis-related functions. Similarly, Wampach et al. found differences in the earliest functional profile according to the delivery mode, including LPS biosynthesis routes being enriched in vaginal deliveries when compared with CS-born neonates [49], which may influence immune system maturation and neonatal health. Despite these observations, these results need to be further evaluated since the prediction of microbiota functionality has been shown to report results that may be not conclusive [50].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDo these differences in the microbiota influence the host immune system response?\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite studies in animal models highlighting the possible effects of microbial colonization patterns on the host gut epithelium maturation and immune system response, little evidence is available from human studies. To the best of our knowledge, this is among the first studies to address this important issue. The intestinal epithelium is the gateway through which gut microbiome\u0026ndash;host crosstalk effects intestinal functionality in the form of enterocytes maturation, mucus production or epithelial barrier development, being a key anatomical location for host\u0026ndash;microbiome interplay.\u003c/p\u003e\n\u003cp\u003eThe samples from HB infants exhibited a higher immune stimulatory capacity than those from hospital-born infants (both VAG and CS), with an increased ability to induce the expression of immune system-related genes (\u003cem\u003eTLR4 \u003c/em\u003eand\u003cem\u003e IRAK\u003c/em\u003e mRNA) and cytokine responses in the HT-29 and THP-1 models, including IL6 and IL8. In concordance with our results, Wampach et al. identified the higher immunostimulatory potential of the microbiota of vaginal-delivered infants when compared with CS-born infants, although they used LPS purified from infant fecal samples and primary human macrophages differentiated into dendritic cells [49]. Combellick et al. noted the higher expression of \u003cem\u003eTLR4\u003c/em\u003e and \u003cem\u003eIL8\u003c/em\u003e mRNA by the HT-29 cell line following exposure to sterile fecal supernatant from HB infants when compared with hospital-born infants. However, they also found mRNA upregulation of the anti-inflammatory cytokine \u003cem\u003eTGF-\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e in hospital-born neonates [34]. We observed that the THP-1 cell line response was more affected by the mode and place of delivery than the HT-29 cell line, which highlighted the importance of the epithelial integrity and the innate immune system on the \u003cem\u003ein vitro\u003c/em\u003e assessment of the host-microbiome interplay.\u003c/p\u003e\n\u003cp\u003eMost prior studies with similar objectives involved acute exposure on unique cell lines. We hypothesized that acute exposure to microbial products could not accurately reflect the biological effect of microbial metabolites and so proposed long-term (7-days) \u003cem\u003ein vitro \u003c/em\u003eexposure assays, including the crosstalk between different cell types, which enabled us to obtain personalized results for each participant, thereby translating the individual signatures to the \u003cem\u003ein vitro\u003c/em\u003e system.\u003c/p\u003e\n\u003cp\u003eIn our model, the HB fecal supernatants induced higher gut barrier integrity (Figure 5) and functionality (IAP; Figure 5) following a time-dependent response that highlighted the relevance of the dynamics of host\u0026ndash;microbiome interplay. Interestingly, the impaired closure of gut mucosal membranes has been shown, alongside higher intestinal permeability, in preterm infants who received formula feeding rather than breastfeeding [51]. This increased permeability could be related to allergic diseases in non-breastfed children [52] and other health disorders [53]. In addition, the HB samples induced the expression of anti-inflammatory molecules (e.g. \u003cem\u003eIL10\u003c/em\u003e, \u003cem\u003eTOLLIP\u003c/em\u003e) to a higher extent than the CS samples, indicating negative feedback on the inflammatory signaling in the gut. Specifically, IL10 down-regulate the microbiota-activated mucosal inflammatory cytokines, reinforce the gut epithelium barrier and control gut permeability, all essential factors to maintaining intestinal homeostasis [54]. Another key element of the innate immune response of the gut is the protective mucus layer covering the epithelium [55]. Higher mucus production was observed after cell-exposure to fecal supernatants obtained from hospital-born infants when compared with HB infants. Both, microbiota [56] and TLR expression [57] are involved in the regulation of mucus production. Despite lower mucus production in the gut being associated with disease phenotypes (e.g. inflammatory bowel disease, higher susceptibility to bacterial infections) [58] in adults, little is known about the role of mucins in neonatal colonization processes. We hypothesize that a penetrable mucus layer in newborns would allow for microbial colonization and interaction with the epithelium during the immune-priming window.\u003c/p\u003e\n\u003cp\u003eMany researchers have discussed the possible relationship between CS and altered immune system development [26]. Generally, CS delivery is associated with the poor stimulation of the immune system [59\u0026ndash;61]. Some researchers have proposed non-diverse environments in early life, including delivery, to play a role in immune maturation and triggering. Furthermore, recent evidence has shown that early exposure to rural areas or farm environments could affect microbial composition and diversity [62], which may be linked to a reduced risk of suffering atopies in adulthood [63]. Kirjavainen et al. recently described how a farm-like indoor microbiota could also decrease the asthma risk in a non-farm environment [64].\u003c/p\u003e\n\u003cp\u003eHowever, most of these results were derived from observational studies, as very few mechanistic analyses have been conducted to date. Our results suggest a possible link between CS and the delayed maturation of intestinal function and the innate immune system. Such a link could play a significant role in the diseases associated with intervention-based deliveries, including autoimmunity, allergy and other immune- and metabolic-related disorders.\u003c/p\u003e\n\u003cp\u003eIn this regard, CS-born infants from our cohort showed higher BMI and W/L z-scores during the first 18 months of life. Other researchers have reported similar results, associating CS with the risk of overweight in children [65,66]. These results could indicate the relevance of priority events in infant health, including those altering microbial colonization, , thereby supporting the early programming hypothesis [67,68]. Researchers have described how antibiotic therapy during early life modulates weight gain in different ways depending on the antibiotic dose in both animal [69] and human epidemiological studies [66]. It has been suggested that high-dose antibiotics can cause important reductions in the microbiota population, which may be related to the weight loss observed in some studies [70]. However, lower antibiotic doses would cause microbiota composition shifts, more than population size variation, and trigger the weight gain shown in the above-mentioned studies. Thus, it remains to be discovered whether the proposed mechanism could be extended to other perinatal factors that also disrupt the microbiota composition and transmission.\u003c/p\u003e\n\u003cp\u003eThe limitations of this study include the low number of participants and the possible confounding factors not included in the analysis (e.g. maternal diet, lifestyle or number of siblings, pets among others). Our microbiota analysis was based on the taxonomic profile obtained via 16S rRNA gene sequencing, which offers less resolution than complete shotgun metagenome sequencing. As we used sterile fecal supernatant, we observed the effects of soluble bacterial metabolites and also, of non-bacteria-related products, including growth factors or eukaryotic extracellular vesicles, which may have influenced the observed results. The use of cell lines may hamper the translational results, although it offers a reproducible and economically viable strategy for further testing on more physiologically relevant models. Among the strengths of the study are the inclusion of three groups and the comparison of CS and vaginal delivery at both hospital and home, including 18 months of follow up. We performed the cellular exposure assays in cellular models with different degrees of complexity and different exposure times, including the epithelial barrier function and maturation as relevant targets, together with the innate immune response. Yet, fecal supernatants contain a complex array of molecules representative of the \u003cem\u003ein vivo\u003c/em\u003e condition, which retain inter-individual differences and features.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur results may provide a mechanistic linkage between studies associating CS and immune-related diseases with colonization pattern alterations, although we cannot rule out other possible factors that might participate in the process. The study has shed light on the effects of hospitalization and HB on neonatal microbial colonization and on the possible effect on innate immune system development, specifically at the intestinal level. The results highlight both the importance of host\u0026ndash;microbial contact during the first month of life and the dynamism of the process. However, further research is needed to determine the impact of these observations in neonatal \u003cem\u003ein vivo\u003c/em\u003e clinical conditions. Such knowledge would facilitate the design of strategies for adjusting medical practices with the aim of reducing intervention during the birth process and ensuring the correct initiation of bacterial colonization and, consequently, the immune system response during early life.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSubjects and Sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA prospective cohort study was conducted to compare the intestinal microbiota of delivered infants born at the hospital (VAG, n=92 and CS, n=65) and at home (HB, n=24). Infants with available biological samples at birth, 7 days and 1 month, together with clinical data, were included.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthical approval for the study was obtained from the Ethics/Bioethics Committee for Clinical Research of Hospital La Fe, Hospital Clinic, Parc de Salut MAR and CSIC (Consejo Superior de Investigaciones Cient\u0026iacute;ficas) [ClinicalTrial.gov NCT03552939].\u003c/p\u003e\n\u003cp\u003ePregnancy, intrapartum variables and anthropometric data were recorded (Additional file 1). Maternal age, maternal pre-pregnancy weight, weight gain over the pregnancy, maternal smoking status, mode of delivery, place of birth (home or hospital), birth weight and length, sex of the neonate, birth instrumentalization, maternal antibiotic exposure during pregnancy and maternal/infant antibiotic use at birth were also collected.\u003c/p\u003e\n\u003cp\u003eInfant length and weight were also registered at birth, 1, 6, 12, and 18 months. Z-scores of anthropometric measures were electronically computed using WHO Anthro software \u003cu\u003e(www.who.int/ childgrowth/software/en/)\u003c/u\u003e\u003cu\u003e.\u003c/u\u003e The WHO Child Growth Standards provide child growth measures standardized by age and sex using z-score.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFecal DNA Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal DNA was extracted from the fecal material (approx. 50-100 mg) using the Master-Pure DNA extraction Kit (Epicentre, Madison, WI, US) following the manufacturer\u0026rsquo;s instructions with the following modifications: samples were treated with lysozyme (20 mg/mL) and mutanolysin (5U/mL) for 60 min at 37\u0026ordm;C and\u0026nbsp;a preliminary step of cell disruption with 3-\u0026mu;m diameter glass beads during 1 min at 6 m/s by a bead beater FastPrep 24-5G Homogenizer (MP Biomedicals). Purification of the DNA was performed using DNA Purification Kit (Macherey-Nagel, Duren, Germany) according to manufacturer\u0026rsquo;s instructions. DNA concentration was measured using Qubit\u0026reg; 2.0 Fluorometer (Life Technology, Carlsbad, CA, US) for further analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing and Bioinformatics Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuality-trimmed and filtering was assessed using DADA2 v. 1.12.1 pipeline [74]. After quality examination, reads were trimmed at the 270th and 210th nucleotide in forward and reverse position, respectively. Additionally, adapters were also removed in the filtering process and a maximum of 2 expected errors was considered. The following denoising and merging steps were performed, and chimeras were also removed. Taxonomic assignment was conducted using the Silva v132 database with the addition of the specie level classification by the same database.\u003c/p\u003e\n\u003cp\u003eTaxa occurring \u0026lt;3 reads in at least 10% of the total samples number and those representing less than 0.01% of the reads across all the samples were filtered. Furthermore, the \u003cem\u003edecontam\u003c/em\u003e package v. 1.4.0 [75] in R environment [76,77] was used to determine the presence of potential contaminants-related sequence. Samples with less than 1000 reads were also removed from the final analysis (n=6). One of those samples was from a vaginally born infant with samples only at delivery time and this infant data was eliminated in further analysis. \u0026nbsp;The final ASV (amplicon sequence variance) table is listed in \u003cstrong\u003eAdditional file 11\u003c/strong\u003e. The 16S rRNA gene sequence data generated is available through NCBI Sequence Read Archive Database under project accession number BioProject ID PRJNA614975.\u003c/p\u003e\n\u003cp\u003ePredictive inferred functional analysis was performed using PICRUST v. 1.1.4 pipeline [78] and the linear discriminant analysis effect sized (LEfSe) analysis was performed for the biomarker discovery using a size-effect cut-off of 3.0 on the logarithmic LDA score [79] using the R code available in the yingtools2 package (\u003ca href=\"https://rdrr.io/github/ying14/yingtools2/man/lefse.html\"\u003ehttps://rdrr.io/github/ying14/yingtools2/man/lefse.html\u003c/a\u003e) and the Dr. Huttenhower\u0026rsquo;s lab galaxy repository of bioinformatic tools.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial Quantification by quantitative PCR Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA small subset of samples (n= 248) according to DNA availability (delivery n=78; 7d n=100; and 31d n=86) were used for the specific bacterial count determination by the qPCR. Total bacterial and \u003cem\u003eBifidobacterium \u003c/em\u003egenus counts were measured by quantitative system based on the amplification of specific 16S rRNA gene region by use of Light Cycler 480 Real-Time PCR System (Roche, Basilea, Switzerland). The (Details in Additional file 11). Reaction mixture consisted in SYBR Green I master mix (Roche, Basilea, Switzerland), 0.25 \u0026micro;M of each specific primer set and 1 ul of DNA. The amplification process consists of one cycle at 95\u0026ordm;C for 5 min, followed by 40 cycles at 95\u0026ordm;C for 20 s, annealing temperature (\u003cstrong\u003eAdditional file 12\u003c/strong\u003e) for 10 s and 72\u0026ordm;C for 10 s. Melting curves were also assessed to test the specificity of the reaction. Standard curves for the specific targeted bacterial group were generated using \u003cem\u003eCt\u003c/em\u003e values and the calculated gene copies numbers were determined based on the fragment amplification length.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the reagents for cell culture were purchased from Sigma-Aldrich, Spain, otherwise stated.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNF-\u0026kappa;B-SEAP HT29 reporter cells\u003c/em\u003e. The HT-29-transfected cell line was previously established in the Laboratory of Lactic Acid Bacteria and Probiotics of IATA-CSIC, by stable transfection of HT-29 cells with a NF-\u0026kappa;B- secreted alkaline phosphatase (SEAP) plasmid (pNiFty2-SEAP; Invitrogen, Carlsbad, CA, US) as a reporter. Cell maintenance was performed in 75 cm\u003csup\u003e2 \u003c/sup\u003eflask with Dulbecco's Modified Eagle Medium (DMEM) High glucose medium supplemented with 1% (v/v) L-Glutamine 200 mM, 1% (v/v) Na-Pyruvate, 1% (v/v) penicillin/streptomycin and 10% (v/v) of inactivated Fetal Bovine Serum (Biowest). Zeocin (200 \u0026micro;g/ml) (InvivoGen) was added to the medium for the clone selection in each passage. All cultures were used between the passage 15 and 20.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTHP-1 cells\u003c/em\u003e. The THP-1 cells were obtained from the European Collection of Authenticated Cell Cultures (THP-1 ECACC 88081201, Public Health England, UK). Cell maintenance was carried out as described in Boudish et al. [80]. All cultures were used between the passage 40 and 50.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCaco-2 and LS17T cells. \u003c/em\u003eThe Caco-2 (ECACC 86010202, Public Health England, UK) and LS174T (ECACC 87060401, Public Health England, UK) cells were obtained from the European Collection of Authenticated Cell Cultures. Caco-2 cells were maintained as described in [81]. LS174T cells were maintained in 25 cm\u003csup\u003e2\u003c/sup\u003e flasks with Eagle's Minimum Essential Medium (EMEM), supplemented with 1% (v/v) GlutaMax, 1% (v/v) Non Essential Amino Acids (NEAA), 10% inactivated Fetal Bovine Serum (FBS) and 1% (v/v) penicillin/streptomycin. Medium was refreshed every two days and cells were subcultured when they reached 80% confluence, as described in [81]. All cultures were used between the passage 40 and 50. Cell morphology was analyzed and checked by phase- contrast microscopy (Olympus CKX41, Olympus Corporation, Tokyo, Japan).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStimulation of HT-29 and THP-1 cell line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of microbial shifts on NF-\u0026kappa;B activation and innate immune response, NF-\u0026kappa;B-HT-29-reporter cells and macrophage-like cell line (THP-1) were exposed to filtered fecal supernatant obtained from a subset of samples of each group (n=4, total n = 12) at 1 month. Samples were randomly selected among the samples with fecal material availability.\u003c/p\u003e\n\u003cp\u003eFecal supernatants prepared as described above (n=4 individuals of each group, total n = 12) were filter-sterilized (0.22 \u0026micro;m PES; Sarstedt SA, Barcelona, Spain) and exposed to the cells as described below. pH adjustment to 7.0-7.2 was performed when required with 0.5M filter-sterilized NaOH (Panreac, Barcelona, Spain) and buffered with HEPES (1% v/v).\u003c/p\u003e\n\u003cp\u003eHT-29 cells were seeded at 7 x 10\u003csup\u003e4\u003c/sup\u003e cells/well in 96-wells plates and incubated for 24h in DMEM High Glucose medium without FBS supplementation. Cells were exposed to filtered fecal supernatant obtained from the three studied groups diluted in 1:10 v/v in DMEM with FBS. Supernatants were collected after 24h of stimulation and SEAP activity was measured using p-nitrophenyl phosphate according to manufacturer\u0026rsquo;s instructions (Thermo Fisher Scientist, Waltham, US). The signal was quantified using a Spectrostar Nano microplate reader (BMG Labtech, Ortenberg, Germany) at 405 nm.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTHP-1 cells were seeded at 5 x 10\u003csup\u003e4\u003c/sup\u003e in 96 wells plates in RPMI 1640, supplemented with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA). After 48 h, cells were refreshed with RPMI without PMA and incubated for 4-5 days, to allow macrophage-like differentiation of THP-1 cells. Thereafter, the cells were exposed fecal supernatants diluted 1:20 (v/v) in RPMI and incubated for 5h. Next, cell culture supernatants were collected, and cells were washed twice with PBS. Cells and supernatants were stored at -80\u0026ordm;C for gene expression determination and cytokine quantification, respectively.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTriple co-culture in Transwell plates: Long-term stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA subset of samples from each group at 1 month of life (n per group=3, total n = 9) were selected to investigate in a more physiologically model the long-term effect of the differences observed in microbiota composition of the infants on the gut barrier and innate immunological state.\u003c/p\u003e\n\u003cp\u003eCell differentiation and the posterior tests were carried out in double chamber wells (Corning\u0026reg; Transwell\u0026reg;-6 well, pore size 0.4 \u0026micro;m; Costar, NY) equipped with separate apical and basolateral compartments and a porous support on which the Caco-2 and LS174T cells grow into a monolayer. The cells were seeded at a density of 7.5 x 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u0026sup2;, in a proportion of 90/10 Caco-2/LS174T in supplemented DMEM. After 24 hours, apical media was refreshed, basal media was removed, and THP-1 cells were seeded in the bottom of the 6 well plate at a density of 1 \u0026middot;10\u003csup\u003e5\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e in RPMI 1640 media containing 100 ng/mL of phorbol 12-myristate 13-acetate (PMA).\u003c/p\u003e\n\u003cp\u003eAfter 48 hours of incubation, both apical and basolateral compartments were refreshed with DMEM or RPMI 1640 without PMA and without antibiotics, respectively.\u0026nbsp; Thereafter, the triple co-culture was maintained for 4 days more and refreshments of the apical and basal media were done every 2 days.\u003c/p\u003e\n\u003cp\u003eFilter-sterilized fecal supernatants selected from each studied group (n=3 individuals, total n = 9) were diluted 1:20 in cell culture media without antibiotic/antifungals and added to the apical compartment of the triple co-culture model (1.5 mL). Treatments were maintained for 7 days, with daily refreshments of the apical and basolateral compartments, with fecal supernatants or RPMI 1640, respectively.\u0026nbsp; Aliquots of the culture supernatant from apical and basolateral media were stored at -80\u0026deg;C for cytokine measurements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEpithelial barrier function: Trans-epithelial electrical resistance (TEER) and apparent permeability.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe monolayer integrity was assessed by measuring the trans-epithelial electrical resistance (TEER) and the apparent permeability (Papp) of the paracellular transport marker Lucifer yellow (LY).\u003c/p\u003e\n\u003cp\u003eA Millicel-ERS (Millipore Corporation, Spain) was used for the TEER measurements. Measurements of the TEER were performed every day at the beginning of the exposure to sterilized fecal supernatant (7 days post-seeding) and every day until the end of the assay (15 days post-seeding). TEER values are reported as delta (\u0026Delta;) of the initial time point before of the exposure and final time point (7 days). TEER values (ohms/cm\u003csup\u003e2\u003c/sup\u003e) are presented in Additional file 9.\u003c/p\u003e\n\u003cp\u003ePapp of LY was measured by adding the marker (100 \u0026micro;M) to the apical compartment of the wells. After 30, 60 and 120 min, 100 \u0026micro;L of medium was removed from the basolateral compartment and replaced with an equal volume of fresh medium (supplemented RPMI-1460 without antibiotics). LY fluorescence was measured at an excitation/emission wavelength of 485/520 nm in 96 black plates (Greiner), using a microplate fluorescence reader CLARIOstar Plus (BMG Labtech, Ortenberg, Germany). A calibration curve (0, 5, 10, 25, 50 and 100 \u0026micro;M) for LY quantification was run in duplicate in each reading. The Papp coefficients were calculated as previously described in [82].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMucus production and Intestinal alkaline phosphatase (IAP) determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt day 15 post-seeding (7 days of fecal supernatant exposure), the transwell inserts containing Caco-2/LS174T cells were incubated with 1 mL of 10 mM of N-acetyl cysteine (Sigma) for 1h at 37\u0026deg;C, 95% humidity in DMEM, adjusted at pH 7-7.2, and the mucus produced by the cells was collected recovering the media and washing once with 0.5 mL of DMEM. The solution containing mucus was concentrated using a Vacuum Concentrator (Eppendorf, Hamburg, Germany) until dryness and re-suspended overnight in 100 \u0026micro;L of PBS at 4\u0026ordm;C. The amount of mucus was measured by a Bradford protein quantification assay, following the manufacturer instructions. Blanks containing cell culture media were subtracted and a standard curve of bovine serum albumin (BSA) was used for the calibration curve (0-5 mg/ml).\u003c/p\u003e\n\u003cp\u003eIntestinal alkaline phosphatase (IAP) activity was assessed in the apical and basal compartment supernatants by enzymatic assay following manufacturer\u0026rsquo;s instructions (Sigma-Aldrich, Missouri, US) scaling the reaction to 100 \u0026micro;l using 4 \u0026micro;l of cell supernatant. Results were read in a SpectroStar Nano (BMG Labtech, Ortenberg, Germany) at 405 nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine quantification in cell supernatant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL6, IL8 and TNF-a released by the cells after acute and long-term exposures to fecal supernatants were quantified by Enzyme-Linked ImmunoSorbent Assay (ELISA), following manufacturer instructions. Human IL8, TNF-a or IL6 Uncoated ELISA kit (Invitrogen, Carlsbad, CA, US) were used for the cytokine determination. In the long-term exposure, cytokine released values were expressed as percentage of variation comparing each treatment to control in order to avoid time-dependent effect. Samples were diluted in assay buffer to adjust the concentration to the linear range of the standard curve.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression by real time RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHT-29 and THP-1 cells from the acute exposure as well as those from tripe co-culture system were collected by scraping at the end of the treatment. RNeasy mini kit (Qiagen, Hilden, Germany) was used to RNA extraction following the manufacturer\u0026rsquo;s instructions. Total RNA was converted to cDNA by Transcriptor First Strand cDNA Synthesis Kit (Roche, Basilea Switzerland) adjusting to the same amount of RNA for each cell type. RT-qPCR analysis was performed using 1 \u0026micro;l of resulted cDNA reaction and 0.25 \u0026micro;M of the specific primers using Lightcycler 480 SYBR Green I master mix (Roche, Basilea, Switzerland). Plates were read in the LightCycler 480 (Roche, Basilea, Switzerland) at annealing temperature of 58\u0026ordm;C. Sequences of the primers used in the study are listed in \u003cstrong\u003eAdditional file 12\u003c/strong\u003e. Genes related with microbes sensing as TLRs and related transcription factors were analyzed and Actin (ACTB) gene was used as housekeeping gene expression, except for the studies in THP1 cell line in the triple co-culture system where hypoxanthine phosphoribosyl transferase (HPRT) gene was used as reference gene for showing higher stability between samples than Actin gene.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChi-Squared test was used to assess differences in the categorical variables of the studied population and ANOVA or Kruskall-Wallis, followed by a Dunn\u0026rsquo;s post-hoc test, was used for the continuous variables according to the distribution of the data. Normality distribution was tested by Shapiro-Wilk test.\u003c/p\u003e\n\u003cp\u003eSequencing data were transformed to relative abundance before further analysis. Pyloseq v. 1.28.0 [83] and vegan v. 2.5-6 [84] packages were used for the analysis conducted in the sequencing data, including alpha diversity estimation. For alpha diversity analysis, samples were rarefied with a 90% of the minimum sample depth. Kruskall-Wallis followed by Dunn\u0026rsquo;s post-hoc tests were performed to detect significant differences in the gut microbial alpha-diversity according to categorical variables studies using vegan v. 2.5-6 and PMCMRplus v. 1.4.4 packages. Spearman correlations were used to find associations between alpha-diversity measures and continuous variables of the population, including gestational age or maternal body mass index, among other, and also between microbial species.\u003c/p\u003e\n\u003cp\u003eFor beta-diversity analysis, Permutational multivariate analysis of variance (PERMANOVA) was conducted to assess the effect of the studied factors on the neonatal gut microbial composition at ASV level (Bray-Curtis distance) and its functionality. For each factor, differences in the dispersion were also tested. In groups with different dispersions, ANOSIM test was also addressed. Calypso web platform v. 8.56 [85] was used for visualizing the multivariate analysis. The clustering of the samples according to the different studied variables, including mode and place of delivery was performed by discriminant analysis of the principal components (DAPC) at ASV level. Calypso platform was also used for Venn diagrams plotting.\u003c/p\u003e\n\u003cp\u003eKruskal Wallis test followed by a Dunn\u0026rsquo;s post-hoc test with the FDR method for multiple comparisons correction was applied to find significant differences in gut microbial composition or functionality (KEGG categories) between studied groups at each time point.\u003c/p\u003e\n\u003cp\u003eFor RT-qPCR analysis, LC480 Conversion version 2014.1 and LinRegPCR v. 11.0 software [86,87] were used for efficiency calculation and gene expression data were analyzed by REST2009 [88]. Statistical analysis of data from in vitro experiments and triple co-culture system was performed by Graphpad software v. 5.04 (GraphPad Software, La Jolla CA, US). Unpaired t-test was used for statistical analysis of mucus production, IAP, TEER and apparent permeability. Kruskall-Wallis and Mann-Whitney test was used for ELISA measurements, considered as non-parametric data. A \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e was considered as a threshold to accept a statistically significant difference. All multiple comparisons were adjusted by false discovery rate (FDR) adjustment method. Each cell culture experiment was performed in triplicate. The R code used in this analysis are available in Additional file 13.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the families involved in MAMI study as well as the contribution of the MAMI team including students, technicians and MDs who participated in obtaining the samples and preparing the metadata. We thank Teresa Gonzalo del Moral and Pepi Dom\u0026iacute;nguez Cano, midwifes from Cooperativa Tit\u0026agrave;nia-Tasc\u0026oacute;; Marga Franch i Ferrer, midwife from Cooperativa Mudra; Concha Delgado and Adela Atero, midwifes from Casa de parts Mitjorn. Asociaci\u0026oacute;n de Comadronas de parto en casa de Catalu\u0026ntilde;a (ALPAC), Barcelona, Spain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC starting grant, n\u0026deg; 639226). M. Selma-Royo is supported by a Pre-doctoral Fellowship from Generalitat Valenciana (GVA)-European Social Fund (ASCII2016).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTORS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors\u0026rsquo; responsibilities were as follows: MCC: designed the study; AP, RE, CM,: recruited the families, managed the collection of the biological samples and clinical data associated; IG was coordinating the cohort information and biological samples processing and metadata; MC and MS were carried out the culture cells models and \u003cem\u003ein vitro\u003c/em\u003e experiments. MS was responsible for the microbiota data and statistical analysis and wrote the first draft. All authors: read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSee ethics paragraph in the \u0026ldquo;Materials and methods\u0026rdquo; section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article is available in the NCBI\u0026rsquo;s Sequence Read Archive (SRA) repository, BioProject ID PRJNA614975 (\u003cbr /\u003e \u003ca href=\"http://www.ncbi.nlm.nih.gov/bioproject/614975\"\u003ehttp://www.ncbi.nlm.nih.gov/bioproject/614975\u003c/a\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of potential conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeBlanc JG, Milani C, de Giori GS, Sesma F, van Sinderen D, Ventura M. 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Normalization of Host Intestinal Mucus Layers Requires Long-Term Microbial Colonization. Cell Host Microbe. NIH Public Access; 2015;18:582\u0026ndash;92.\u003c/li\u003e\n\u003cli\u003eBhinder G, Stahl M, Sham HP, Crowley SM, Morampudi V, Dalwadi U, et al. Intestinal Epithelium-Specific MyD88 Signaling Impacts Host Susceptibility to Infectious Colitis by Promoting Protective Goblet Cell and Antimicrobial Responses. Infect Immun. American Society for Microbiology (ASM); 2014;82:3753.\u003c/li\u003e\n\u003cli\u003eSchroeder BO. Fight them or feed them: How the intestinal mucus layer manages the gut microbiota. Gastroenterol. Rep. Oxford University Press; 2019. p. 3\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eHuurre A, Kalliom\u0026auml;ki M, Rautava S, Rinne M, Salminen S, Isolauri E. Mode of Delivery \u0026ndash; Effects on Gut Microbiota and Humoral Immunity. Neonatology. 2008;93:236\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eNikischin W, Peter M, Oldigs HD. 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MAMI: a birth cohort focused on maternal-infant microbiota during early life. BMC Pediatr. 2019;19:140.\u003c/li\u003e\n\u003cli\u003eKlindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013;41.\u003c/li\u003e\n\u003cli\u003eBolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. Oxford University Press; 2014;30:2114\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eCallahan BJ, McMurdie PJ, Rosen M, Han A, Johnson A, Holmes S. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003eDavis NMN, Proctor D, Holmes SSP, Relman DA, Callahan BJ. Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. bioRxiv. 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Oxford University Press; 2017;33:782\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003eRuijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37:e45\u0026ndash;e45.\u003c/li\u003e\n\u003cli\u003eRamakers C, Ruijter JM, Deprez RHL, Moorman AFM. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett. 2003;339:62\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003ePfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. Oxford University Press; 2002;30:e36.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Additional Files","content":"\u003cp\u003e\u003cstrong\u003eAdditional file 1.\u003c/strong\u003e Characteristics of studied population according to place and mode of delivery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2.\u003c/strong\u003e Neonatal fecal microbiota diversity and richness of meconium and infant fecal samples at 7 and 31 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 3.\u003c/strong\u003e Relative abundance of neonatal fecal microbiota along the first month of life\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 4. \u003c/strong\u003eTaxonomic biomarkers of microbiota composition of each group depending on place and mode of delivery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 5. \u003c/strong\u003eCore group of neonatal microbiota composition at genus level over the first moth of life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAddition file 6. \u003c/strong\u003eQuantitative analysis of intestinal microbiota from infants born at hospital (vaginal and C-section delivery) and at home across the first month of life\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 7. \u003c/strong\u003eMicrobial functions related to amino acids metabolism computationally predicted present in neonatal microbiota along the first month of life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 8.\u003c/strong\u003e Gene Expression of HT-29 and THP-1 cells after 24 h of fecal supernatant exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 9.\u003c/strong\u003e Epithelial barrier function and maturation of simulated intestinal epithelium of the triple co-culture during the long-term exposure\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 10.\u003c/strong\u003e Flow chart of study participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 11.\u003c/strong\u003e Amplicon sequence variant (ASV) table.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 12. \u003c/strong\u003ePrimers of 16s rRNA gene of prokaryotic targets and human genes tested by qPCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 13.\u003c/strong\u003e R code used in the study and links of consulted GitHub repositories\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Microbiota, environment, mode of birth, antibiotics, epithelial barrier, immune system, early programming","lastPublishedDoi":"10.21203/rs.3.rs-20279/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-20279/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Early microbial colonization triggers processes that result in intestinal maturation and immune priming. Perinatal factors, especially those associated with birth, including both mode and place of delivery are critical to shaping the infant gut microbiota with potential health consequences. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Gut microbiota profile of 180 healthy infants (n=23 born at home and n=157 born in hospital, 41.7% via caesarean section [CS]) was analyzed by 16S rRNA gene sequencing at birth, seven days and one month of life. Breastfeeding habits, infant clinical data, including length, weight and antibiotic exposure, were collected up to 18 months of life. Long-term personalized \u003cem\u003ein vitro\u003c/em\u003e models of the intestinal epithelium and innate immune system were used to assess the link between gut microbiota composition, intestinal function and immune response. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Microbiota profiles were shaped by the place and mode of delivery, and they had a distinct biological impact on the immune response and intestinal function in epithelial/immune cell models. Bacteroidetes and \u003cem\u003eBifidobacterium\u003c/em\u003e genus were decreased in C-section infants, who showed higher z-scores BMI and W/L during the first 18 months of life. Intestinal simulated epithelium had a stronger epithelial barrier function and intestinal maturation, alongside a higher immunological response (TLR4 route activation and pro-inflammatory cytokine release), when exposed to home-birth fecal supernatants, compared with CS. Distinct host response could be associated with different microbiota profiles. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Mode and place of birth influence the neonatal gut microbiota, likely shaping its interplay with the host through the maturation of the intestinal epithelium, regulation of the intestinal epithelial barrier and control of the innate immune system during early life, which can affect the phenotypic responses linked to metabolic processes in infants.\u003c/p\u003e","manuscriptTitle":"Perinatal Environment Shapes Microbiota Colonization And Infant Growth: Impact On Host Response And Intestinal Function","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-07-10 20:15:16","doi":"10.21203/rs.3.rs-20279/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-10-14T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-07-09T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-07-08T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-07-08T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-04-01 16:25:59","doi":"10.21203/rs.3.rs-20279/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-06-25T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-06-18T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-04-28T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-04-21T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-04-18T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-04-18T12:00:00+00:00","index":1,"fulltext":""},{"type":"submitted","content":"","date":"2020-03-30T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-03-30T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-03-29T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-03-29T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e58e92fd-32a0-4d25-b15d-9006465b0e15","owner":[],"postedDate":"July 10th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":149872,"name":"General Microbiology"}],"tags":[],"updatedAt":"2020-11-29T15:03:42+00:00","versionOfRecord":{"articleIdentity":"rs-20279","link":"https://doi.org/10.1186/s40168-020-00940-8","journal":{"identity":"microbiome","isVorOnly":false,"title":"Microbiome"},"publishedOn":"2020-11-23 15:02:19","publishedOnDateReadable":"November 23rd, 2020"},"versionCreatedAt":"2020-07-10 20:15:16","video":"","vorDoi":"10.1186/s40168-020-00940-8","vorDoiUrl":"https://doi.org/10.1186/s40168-020-00940-8","workflowStages":[]},"version":"v2","identity":"rs-20279","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-20279","identity":"rs-20279","version":["v2"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}