Acquisition of microbiota according to the type of birth: an integrative review

Objective: to analyze scientific evidence regarding the relationship between the type of birth and the microbiota acquired by newborns. Method: this integrative review addresses the role of the type of delivery on newborns’ microbial colonization. A search was conducted in the Medical Literature Analysis and Retrieval System Online/PubMed and Virtual Health Library databases using the descriptors provided by Medical Subject Headings (MeSH) and Health Science Descriptors (DeCS). Results: infants born vaginally presented a greater concentration of Bacteroides, Bifidobacteria, and Lactobacillus in the first days of life and more significant microbial variability in the following weeks. The microbiome of infants born via C-section is similar to the maternal skin and the hospital setting and less diverse, mainly composed of Staphylococcus, Streptococcus, and Clostridium. Conclusion: the maternal vaginal microbiota provides newborns with a greater variety of colonizing microorganisms responsible for boosting and preparing the immune system. Vaginal birth is the ideal birth route, and C-sections should only be performed when there are medical indications.


Introduction
The Brazilian obstetrical context is of concern and considered a public health problem due to various factors, including the C-section epidemic. According to the World Health Organization (WHO), the rate of C-sections should not exceed 15% (1) ; however, approximately 90% of the deliveries in the supplementary health system are C-sections, while in the Unified Health System (SUS), this rate reaches 45% (2) .
The route of birth delivery may also influence a newborn's health. Children born via C-section are at increased risk of developing asthma, systemic connective tissue disorders, juvenile arthritis, inflammatory bowel disease, immune deficiencies, and leukemia (9) . Part of these diseases is believed to be related to the maturation of the neonatal immune system (10)(11) .
Studies suggest that the immune system of a newborn is widely stimulated when first exposed to microorganisms during neonatal life (11) , while the type of delivery shapes an infant's microbial communities, which consequently play a role in his/her immune system maturation (12) .
Therefore, we propose that the route of birth delivery influences the colonization of microorganisms in newborns.
Nonetheless, studies seldom address the mechanisms involved in this adaptation according to the type of birth.
This study's findings are expected to support the choice of the type of birth, decreasing unnecessary C-sections without a medical indication. Fetal morbidity and mortality caused by C-sections and inappropriate adaptation of the neonatal immunological system are also expected to decrease. Therefore, this study's objective was to analyze scientific evidence concerning the relationship between the route of birth delivery and the newborns' acquisition of microbiota.

Method
This is an integrative review, which can be defined as a method capable of synthesizing scientific knowledge regarding a given problem (13) . It also allows researchers to monitor a topic's development over time and formulate new theories and generate knowledge (14) . It allows incorporating studies with the most diverse methodological designs, and for this reason, is considered a complex tool (14) .
This study strictly followed the proposed stages (15) , namely: 1st) establishment of the topic and guiding question; 2nd) establishment of inclusion and exclusion criteria; 3rd) identification of studies; 4th) categorization of studies; 5th) analysis and interpretation of results; 6th) synthesis of knowledge and presentation of results.
PICO, which stands for Patient, Intervention, Comparison, and Outcomes, was the strategy used to establish the guiding question. These four components are essential to establish the guiding question and, consequently, seek scientific evidence (16) . Therefore, the topic chosen was the acquisition of microbiota among newborns according to the route of birth delivery. The guiding question was: What is the production of knowledge concerning the association between the acquisition of microbiota among newborns and the route of birth delivery? The first element of the strategy (P) refers to newborns; the second (I) to C-section; the third refers to vaginal delivery (C); and the fourth element (O) refers to newborns' acquired microbiota.
Inclusion criteria were: papers published in Portuguese, English, or Spanish; answering the guiding question; with up to ten years since publication. Exclusion criteria were letters, editorials, experts' opinions, reviews, and studies addressing premature infants, or not comparing the route of birth nor addressing newborns.

The Preferred Reporting Items for Systematic
Reviews and Meta-Analyses -PRISMA was adapted to guide this review's report (18) . After gathering the studies identified in the two electronic databases, those that appeared more than once were excluded after comparing titles, authors, year of publication, and country of origin. The studies were then analyzed using an instrument to collect data (19) addressing the study's title, The studies were also analyzed according to the level of evidence. In this study, the level of evidence was organized into seven levels: Level I -systematic review or meta-analysis of randomized controlled clinical trials; Level II -evidence obtained from at least one well-designed randomized controlled clinical trial; Level III -well-designed controlled trial without randomization; Level IV -evidence from well-designed case-control or cohort studies; Level V -systematic reviews of descriptive and qualitative studies; Level VI -evidence form a single descriptive or qualitative study; Level VII -evidence from the opinion of authorities and/or reports of expert committees (20) .
The studies were tabulated and rigorously analyzed, interpreted, and synthesized. Two thematic categories were chosen according to the topics addressed in the studies to interpret the results.

Results
This study's sample was composed of 25 studies.
The entire process of searching, excluding, and selecting studies is described in detail in a flowchart ( Figure 1).  The vaginal delivery group has significantly more Bifidobacteria and Akkermansiaceae, showing the beneficial aspect of natural birth. Two common pathogenic bacteria, Providencia and Gardnerella, were also found in some of the infants in this group, which may be explained by the presence of mothers with vaginal infections that had not been manifested.

*VHL = Virtual Health Library
Shi, et al. (24) 2018 The microbiome of children born vaginally is a little more diverse than that of children born via C-section. Chu, et al. (27) 2017 The neonatal microbial community structure at the time of delivery did not show significant differences according to the body's site. Vaginal delivery: Lactobacillus; C-section: Propionibacterium and Streptococcus. The microbiota of newborns born vaginally tended to be similar to their mothers' vagina, while infants born via elective C-sections were mainly populated by the microbiota found on their mothers' skin.
Hill, et al. (28) 2017 The structure of an infant's gut microbiota is affected by the type of delivery. Vaginal birth: Bifidobacteria, Bacteroides; C-section: Clostridium. A large diversity of individual population structures was found within each group, showing the heterogeneous gut microbiota composition of developing infants.
Bokulich, et al. (29) 2016 Well-designed controlled trial without randomization/III The children born via C-section showed a significantly greater phylogenetic diversity (p<0.05). However, these significantly decreased during the first month after birth while these children subsequently presented a lower diversity and less richness up to 2 years of age.
Bosch, et al. ( Well-designed controlled trial without randomization/III Regardless of the body site, the microbiome of infants born vaginally or via C-section were more similar to their mothers' vaginal microbiome, when exposed to vaginal fluid, than that of infants born via C-section but not exposed. Kristensen; Herinksen (32) 2016 Cohort/IV C-section was associated with infection and inflammation of the mucosa. The effect of elective C-sections was more significant for asthma than emergency C-sections. Estimates of respiratory tract disease did not change after adjusting for neonatal respiratory morbidity.

Authors Year Study design/Level of evidence Main Results
Martin, et al. (33) 2016 Cross-sectional/VI The type of delivery was one of the factors that strongly impacted the initial composition of newborns' microbiota. The infants born vaginally had significantly higher bacterial counts in the meconium than those born by C-section. Dogra, et al. (37) 2015 Cohort/IV The bacteria colonizing newborns' bodies promote a long-lasting effect in the immune system or on the gut barrier function, driven by the type of delivery. Vaginal birth: Bifidobacterium and Collinsella. C-section: Klebsiella, Enterobacteriaceae, and Streptococcus. A lower or later colonization by Bifidobacteria was found among infants born via C-section; Bifidobacteria are considered to be ideal in a newborn's organism.
Dong, et al. (38) 2015 Cross-sectional/VI The route of the birth delivery had a more significant impact on the gut microbiota structure than on its diversity during the first 4 days of life. Well-designed controlled trial without randomization/III A lower microbial diversity was found among infants born via C-section and lower circulating levels of the Th1-type chemokines, CXCL10 and CXCL11.
Makino, et al. (41) 2013 Well-designed controlled trial without randomization/III The number of Bifidobacteria was significantly lower among infants born through C-section than among infants born vaginally up to 7 days of age. Bifidobacteria gut colonization is considered to begin more rapidly among infants born vaginally than among infants born through C-sections.

Pandey, et al. (42) 2012
Well-designed controlled trial without randomization/III The initial colonization and acquisition of gut microbiota may strongly influence the status of cellular and humoral elements of the immune system of the gut mucosa during a newborn's life.  reflecting on the population's health (23,(45)(46) .
Therefore, it is essential to understand the importance of the microbiota for the immune system and its relationship with the route of birth delivery and a newborn's microbiota composition.
The neonatal period is considered a critical window in the development of the immunological system.
Appropriate microbial stimulation is essential for the appropriate maturation of TReg cells response (55) . Shortchain fatty acids, a by-product of bacterial fermentation, are known for modulating T cells' regulatory homeostasis (TReg) (56) . These are responsible for immunomodulatory or immunosuppressive responses; that is, they play a role in the body's immune tolerance, controlling inflammatory responses (57) . Dysregulation of these cells is directly linked to the development of allergic problems and autoimmune diseases (42,48) .
Additionally, the human organism's microbiota limits the colonization of pathogens based on a competition for metabolites, a process known as "colonization resistance" (57)(58) . The colonizing microbiota establishes a homeostatic relationship with the host, in which the colonization microorganisms benefit from the nutrient-rich environment, ensuring their survival and providing their hosts with a greater ability to absorb nutrients from food, establishing networks and biofilms capable of protecting the organism from pathogenic antigens, among other benefits (45,48) .
Bifidobacterium and Lactobacillus are considered the ideal bacteria for the human body because they are commensal bacteria (22,44,52,59) . The early presence of Bifidobacterium between one week and three months of age was associated with a lower risk of developing eczema (59)(60) . Additionally, a decreased number of Bifidobacterium and Lactobacillus seem to be related to the emergence of allergies (42,52,59) . Some studies do not report significant differences between the microbiota of asthmatic and healthy individuals; however, low levels of Bifidobacterium are found in the long run among individuals with asthma, compared to recently diagnosed individuals (61) .
Another component found in the microbiota of newborns is Bacteroides. Some of them help regulate gut immunity, and a deficiency of these bacteria was identified among infants born via C-section. This situation causes the rupture of tolerogenic response, contributing to inflammation and obesity (37,42) . Additionally, these bacteria are involved in the digestion of human milk, and scarcity of these in the neonatal gut may cause digestive problems (26,28) .
These claims are explained based on the Old Friends theory. Commensal microorganisms train the immunological system to develop tolerance mechanisms as a strategy for their own survival. Hence, the immune system recognizes and eliminates harmful bacteria but does not react against useful species or the organism itself (60,62) . Old Friends bacteria can activate the production of anti-inflammatory cytokines, differentiating themselves from pathogens that promote pro-inflammatory cytokines (52,62) .
Regarding the route of birth delivery and neonatal microbiota, the first influences the acquisition and colonization of bacteria in newborns' bodies 52,63) , especially during early childhood, usually stabilizing in the first year of life (35) . An infant's immune system is exposed for the first time to microorganisms during vaginal delivery or C-section (12) .
Infants born vaginally have a higher concentration of Bacteroides, Bifidobacterium, and Lactobacillus in the first days of life, and a greater microbial variability occurs in the following weeks (22,33,43,47,62) . These infants' microbiota is more similar to their mothers' vaginal microbiome (21)(22)27,44,64) . Note that the main bacterial communities found in mothers' vaginas are Lactobacillus, Bifidobacterium, and Streptococcus (65)(66) , though there are controversies in the literature. The vaginal flora composition during pregnancy is relatively less diverse compared to that of non-pregnant women. This is due Coelho GDP, Ayres LFA, Barreto DS, Henriques BD, Prado MRMC, Passos CM.
to a decreased number of some members of the vaginal community and enrichment of Lactobacillus target species.
Hence, the composition of a pregnant woman's vaginal microbiota is more stable, conferring a protective role against ascending infections (65) .
Infants born via C-section present a microbiome similar to the maternal skin and the hospital environment (47,56,(62)(63) , which is also less diverse, mainly composed of Staphylococcus, Streptococcus, and Clostridium. Although, a longitudinal study (29) addressing 43 infants reports a significantly greater bacterial diversity among those born via C-section than those born vaginally.
This variety, however, decreased from the first month of life up to two years of age while these children presented less phylogenetic richness compared to children born vaginally (29) .
The Staphylococcus, Clostridium, Klebsiella, Enterobacter, and Enterococcus species were more frequently found among infants born via C-section.
These bacteria are resistant to various antibiotics and are endemic in the hospital setting (22)(23)(25)(26)35,44) . Studies suggest that C-sections cause effects similar to the use of antibiotics, changing the maturation patterns of microbiota in neonates (29) .
On the other hand, the effects of elective and emergency C-sections differ (35) . Elective C-sections imposed a greater risk of asthma than emergency C-sections (32) . Infants born via emergency C-section undergo the effects of labor and, for this reason, have a bacterial composition similar to that of infants born vaginally (12) . Consequently, these children are less likely to develop an immune system problem compared to infants born via elective C-sections.
The microbiota of healthy adults is stable and diversified (67) . Aberrant and scarce microbiota are related to various health disorders (40,42,52,54) . Additionally, the low diversity of colonizing bacteria is related to a larger number of antibody-secreting cells, causing exaggerated immune responses (67) . In this sense, the immune system is believed to have a more beneficial response to the  immune systems and its implications in adulthood.