Immune system is crucial to recognize and exclude antigenic foreign particles and maintain the stability of the internal environment. The human immune system begins to develop early in fetal development and has obvious sensitivity to external antigens (1–3). The fetal immunity is likely influenced by fragments and metabolites of maternal gut microbes, whereas the presence of the microbiome in utero has been a controversial topic (4). Earlier reports have shown that the amniotic cavity and placenta are sterile (5–10). The superior defenses of placenta mean it’s extremely difficult for microbes to enter the uterine environment.

It is widely known that the placenta is an essential organ for material exchange and the primary barrier between the mother and the fetus during human pregnancy. The placenta composed of amnion, villous trees and decidua basalis, has the function of defense, synthesis and immunity. Anchoring villi, an integral part of the villous tree, are attached to decidua basalis by extravillous trophoblasts (EVTs). Fetal blood passes through the umbilical artery to the villous capillaries and exchanges material with maternal blood in the intervillous space, but fetal blood and maternal blood are not directly connected. The villous trees of placenta at full term are covered by syncytiotrophoblast, and there is a layer of cytotrophoblasts below which is discontinuous. The inner layer of cytotrophoblasts layer is the basement membrane, which acts as the placental barrier (11). In addition, syncytiotrophoblasts and cytotrophoblasts both provide effective protection against viral and non-viral pathogens. Among them, the surface of syncytiotrophoblast has unique physical properties, and the physical barrier formed by syncytiotrophoblast limits the vertical transmission of pathogens at multiple stages of pregnancy (12–16).

However, with the development of microbial detection technology, more and more evidence suggest the presence of microbes in human placenta and fetus (17–26). Studies supporting the sterile womb hypothesis suggest that the microbial signals detected in the womb are actually due to contamination of samples and the DNA purification kits (21, 27). Researches supporting the presence of a low biomass placental microbiome suggest that after filtering out contaminants and low-quality sequences according to negative controls, some microbial signals still exist (26, 28, 29). Excitingly, a recent study showed that microbial exposure reduces fetal immune cells early in human development. This study demonstrated the presence of microbes in fetal organs by inoculating fetal tissue in culture media and visualizing fetal guts, and suggested that these bacteria induce the activation of syngeneic memory T cells in fetal mLN T cells. And the question of how do microbes get into the uterine environment, has it been shown that pathogens (Zika virus, Toxoplasma gondii, HIV, Cytomegalovirus, etc.) might target multiple cells in the decidua to reach the extravillous trophoblasts (EVTs) layer and eventually bypass the syncytial layer (13, 30, 31). Microbes might use this mechanism to breach the placental barrier, but are more likely to evolve distinct strategies at different stages of pregnancy (11).

Most of the recent studies on fetal microbes are based on 16S rRNA gene amplicon sequencing and metagenomic sequencing (10, 26, 32, 33). Metagenomic data includes bacterial, archaear, protozoa, virus, fungus and host genomes. Compared to 16S rRNA sequencing, the DNA used for metagenomic sequencing is not amplified by PCR, and the metagenomic results are relatively unbiased. Besides, the composition, abundance and function of microbiota can be obtained by metagenomic sequencing (34). However, due to the low biomass of fetal samples, it is difficult to detect archaea and virus signals using 16S rRNA and metagenomic sequencing. Virome is a combination of metagenomic theory and existing virus molecular biological detection technology, mainly used for the studies of all viruses genetic material in the environment (35). Therefore, in order to detect as many microbial signals as possible, virome sequencing were used in this study to explore the fetal gut microbiome based on the metagenomic results.

The presence of microbes in fetal gut has long been controversial. After strict experimental conditions and environmental control settings, our results showed that there were indeed microbes in fetal gut, including bacteria, archaea and viruses.

The integration results of metagenomics and virome showed that Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes were the dominant phyla of fetal gut at the phylum level. At the genus level, Pseudomonas, Bradyrhizobium, Microbacterium, Burkholderia and Ralstonia were the dominant genera of fetal gut, of which Ralstonia and Burkholderia were detected in the environmental control groups in recent studies (26). However, Ralstonia insidiosa, as a member of Ralstonia, was a resident at the maternal-fetal interface in another research (45). Indeed, we also detected a high abundance of Ralstonia pickettii and Ralstonia insidiosa in fetal gut. Our results confirm that a recent study by Mishra et al. analyzed fetal microbes and found that Pseudomonas and Bradyrhizobium were enriched in fetal samples (26).

Besides, we detected 700 species viruses, which together with other gut microbial communities maintain the dynamic balance of gut and are key players in the regulation of intestinal homeostasis and inflammation, including 130 species of bacterial viruses (bacteriophages) and 570 species of eukaryotic viruses in fetal gut. (46, 47). Among the 570 species of eukaryotic viruses, Human betaherpesvirus 5 (also termed human cytomegalovirus) was the most abundant, which is a common cause of congenital viral infection in fetuses and neonates and a major non-genetic cause of congenital sensorineural hearing loss and neurological disability (48, 49). Humans are the only host of human cytomegalovirus (HCMV), which can replicate in most types of cells. HCMV can lead to infection in the developing fetus through vertical transmission during maternal infection (50, 51), and the transmission rate in the first, second, and third trimesters are 26%, 28%, and 65% (52–55). Among the 198 species of bacteriophages that can interact with bacteria to regulate bacterial composition, phages of Clostridium, Escherichia and Flavobacterium were the predominant species. Moreover, we detected crAssphage, which is not only the most abundant virus known to exist in humans but also almost ubiquitous (56), suggesting that crAssphage was acquired in early life. This was contrary to the research from Lim et al. (57).

Most studies currently focused on bacteria, fungi or virus, while archaea are often overlooked. However, the interaction between archaea and host can affect the host in many ways, as archaea were proposed to use for the prevention of trimethylaminuria and cardiovascular disease (58); archaea found on human skin may be related to age and skin physiology (59); archaea participate in the pro-inflammatory process (60). Methane-producing archaea were the predominant component of the archaeome, including Methanobacteriales and Methanomassiliicoccales (61). Methanobrevibacter smithii is the most abundant methanogen in the human gut and was isolated as the first representative nearly 40 years ago (62). Take the advantage of virome sequencing, 262 archaeal species (including 8 phyla and 117 genera) were detected in fetal gut. Euryarchaeota was the most abundant phylum, which contained most of the species of archaea (Methanogens, halophiles and Thermophiles). Methanobrevibacter smithii was the most abundant specie, and this is the first ever detection in fetal gut of Methanobrevibacter smithii, which confirmed the hypothesis from Sereme et al. that Methanobrevibacter smithii was an in-utero member of gut microbiota (63). Our results opposed the hypothesis that breast milk is the source of Methanobrevibacter smithii in premature neonates (64). The study from Grine et al. showed that Methanobrevibacter smithii was detected in vaginal fluid only in cases of vaginal disease (65), although the fetus passed through the vagina, the mother of our study without bacterial vaginosis. Besides, more methanogens such as Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanobrevibacter ruminantium and Methanosphaera stadtmanae were detected in fetal gut.

Both metagenomic and virome sequencing results showed that rectal microbial species were higher than those of small intestine and cecum in different classification levels (phylum, genus and specie). Besides, the composition and functional pathway of the small intestine and rectum were more similar. The difference between the cecum and the other two sites might be due to the host contamination of the cecum was more severe than that of the small intestine and rectum.

We compared the fetal gut microbiota to the adults’ and found that the diversity of adults’ gut microbiota was significantly higher than that in fetal group. The composition of gut microbiota between two groups was significantly different. Specifically, Proteobacteria and Actinobacteria were the dominant bacteria phyla in fetal gut. While the dominant bacteria phyla in adults’ gut were Firmicutes and Bacteroidetes, which indicated that Firmicutes and Bacteroidetes gradually replaced Proteobacteria and Actinobacteria as the main force of gut microbiota during the process of growth and development. For viruses and archaea, the dominant phyla in both groups were similar. Besides, we found 6 ARGs in fetal group were consistent with adult group, suggesting that microbes carried ARGs might transmit vertically during pregnancy.

In conclusion, we detected a variety of microbes in fetal gut through metagenomic and virome sequencing, including bacteria, virus and archaea. Especially, we first identified Methanobrevibacter smithii in fetal gut, which was the most prevalent and abundant methanogen. In addition, by comparing the fetal and adults’ gut microbiota, we found that gut bacterial composition changed greatly during the growth and development process and the same ARGs existed in fetuses and adults. Thus, we suggest the fetal gut is not a sterile environment and has a low abundance but metabolically rich microbiome. Our study provided valuable resource for understanding human fetal immunity development during gestation.

The data presented in the study are deposited in the Genome Sequence Archive for Human repository, accession number: HRA003676.

The studies involving human participants were reviewed and approved by West china 2nd university hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

Experimental work: GH, AL, JL and MH. Data analysis: YL, XYL and WG. Writing and editing: XL, XHL, ZF and YZ. All authors contributed to the article and approved the submitted version.