**3. Discussion**

Advances in molecular methodology reveal the details of the human-microbial relationship, allowing for increased identification of microbiota composition and function. Recently, the maternal microbiome has been shown to prepare the newborn for host– microbial symbiosis, driving postnatal innate immune development [28]. However, the viability of placental bacteria cannot be determined due to discrepancies with the culture results [24]. On the other hand, some of these microorganisms may not be easily cultured, but they can be detected by DNA analysis.

The results of a review evaluating the microbiological composition of the placenta in a healthy pregnant woman and the potential relationship between the placental microbiome and the oral microbiome have shown the existence of a low biomass placental microflora in pregnant women with a normal course of pregnancy [29].

In turn, animal studies have shown that despite differences in gut physiology and morphology, both humans and cattle require a functional microbiome early in life (preimplantation and organogenesis) and throughout pregnancy [25]. Studies indicate that both species acquire intestinal microbes before birth, possibly from the mother, which would indicate the existence of similar mechanisms and timing of fetal intestinal colonization.

Other studies have shown that gut microbiota dysbiosis is an important etiology of pre-eclampsia (PE) [30,31]. The intestinal microbiota and its active metabolites have great

potential in the treatment and diagnosis of PE. The results of the cited work enrich the theory of the enteroplacental axis and contribute to the development of microecological products for preeclampsia. PE and FGR are placental-mediated disorders, and metabolomic studies of maternal-fetal pairings may aid in understanding their pathogenesis. Microbiome profiles from 37 overweight and obese pregnant women enrolled in the SPRING cohort were examined by 16SrRNA sequencing [32,33]. Consistent with our findings, four main bacterial phyla (*Firmicutes*, *Bacteroidetes*, *Actinobacteria* and *Proteobacteria*) were identified in all microbiomes. The possible origin of the placental microbiome was both the maternal oral and gut.

FGR is a complex obstetric complication with various causes and a wide spectrum of complications, especially for the fetus, as it is associated with an increased risk of perinatal mortality and morbidity. As highlighted above, the pathogenesis of FGR is unclear, which limits its effective treatment. It has been found that the dysbiosis of the intestinal microflora plays an important role in the pathogenesis of various diseases. However, its role in the development of FGR remains unclear and requires clarification.

In our study, significantly higher in the study group were bacteria: *Actinopolyspora erythraea*, *Listeria costaricensis*, *E. coli*, Methylobacterium, *Acidobacteria bacterium*, *Bacteroidetes bacterium*, *Paenisporsarcina* sp., *Thiodiazotropha endoloripes* and *Clostridiales bacterium*. On the other hand, in the control group, statistically more frequently, based on proteomic data, the following were found: *Flavobacterial bacterium*, *Aureimonas* sp. and *Bacillus cereus*.

Correlations between, e.g., *Helicobacteria pylori*, and the development of FGR in a group of 600 women were demonstrated by den Hollender et al. [34]. In turn, the important factor, which is the intestinal microbiome of infants, is indicated by the results of research by Groer et al. and Yang et al. [35]. Yang's research has shown correlations between an infant's physical development and fecal cysteine concentrations [36]. It also turned out that *Oscillospira* and *Coprococcus* are involved in the synthesis of butyrate, which is a source of energy for intestinal epithelial cells. Consistent with our results, a study by Tu et al., evaluating the feces of infants with FGR, showed significant differences in the growth of *Bacteroides*, *Faecalibacterium* and *Lachnospira* in patients with growth restriction [37].

In a pilot study by Hu et al., the relationship of FGR with the reproductive microbiome has been studied [38]. The reproductive microbiome was studied by 16sRNA sequencing (20-IUGR, 20-controls). Microbiological screening of the placenta showed a diverse flora as in our results, mainly *Proteobacteria*, *Fusobacteria*, *Firmicutes* and *Bacteroidetes*. The study group with FGR was characterized by a higher incidence of β-hemolytic bacteria *Neisseriaceae* and an increase in the number of anaerobic bacteria *Desulfovibrio* reflective of placental hypoxia. Further analysis of the reproductive microbiome of the FGR samples revealed lower levels of H2O2-producing *Bifidobacterium* and *Lactobacillus* that go from respiration to fermentation, a less energetic metabolic process as oxygen levels drop. Source tracing analysis showed that placental microbial content was predominantly from an oral source, compared to an intestinal or vaginal source. The cited results suggest that reproductive microbiome profiles may be potential biomarkers for fetal health during pregnancy in the future, while *Neisseriaceae* may represent promising therapeutic targets for the treatment of IUGR.

The *Actinopolyspora erythraea* protein identified in our FGR placentas catalyzes the circularization of gamma-N-acetyl-alpha, gamma—diaminobutyric acid (ADABA) to ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid), an effective osmoprotectant [39]. This prokaryote occupies an "extreme or inhabitable environment" [40]. These bacteria (extremophiles) have evolved to harsh pH, temperature, salinity and pressure by biosynthesizing unique compounds, such as new enzymes. *Acidobacteria* appear to be able to resist numerous pollutants, such as PCBs and petroleum compounds, linear alkylbenzene sulfate, p-nitrophenol, and heavy metals, under low pH circumstances [41]. A high number of acidobacterial genes code for transporters belonging to the drug/metabolite transporter superfamily. Unfortunately, no data supporting real actions linked to pollution degradation have been documented. The role of these bacteria in the pathophysiology of FGR is unknown.

*Methylobacterium* was also identified in FGR placentas; it is an emerging opportunistic premise plumbing pathogen (OPPsP) [42]. It possesses chlorine resistance, biofilm development, desiccation tolerance, and resilience to temperatures above 50 degrees Celsius. *Methylobacterium extorquens*, like other OPPPs, was isolated from amoebae in drinking water systems, making it an amoeba-resistant bacteria.

The sophisticated methods used in our research are based on the identification of proteins using LC-ESI-MS/MS by pooling material from 18 FGR and 18 control placentas. To distinguish between placental samples and contamination introduced during DNA extraction, purification and amplification, unsupervised ordination methods showed a separate clustering between pooled negative control and placental samples like in studies performed by others [32]. These methods do not distinguish between live, dead or ruptured bacterial fragments. Differences in relation to the data of other researchers may be statistically significant because the statistical analysis does not concern individual cases but material from the studied population, which seems to be more convincing in terms of the population.

The clinical implication of our research could be a careful consideration in the rational prescription and use of antibiotics to avoid infections while at the same time protecting the fetus from the adverse effects of pharmacotherapy.

A limitation of our study would be that we did not perform any bacterial culture of the tested bearings due to the presence of potentially viable bacteria. The material for the study was collected during cesarean section in sterile conditions. However, other researchers have confirmed that this method is devoid of the possibility of contamination [43]. Moreover, none of the taxa of bacteria mentioned in other studies were found to have different abundance between vaginally delivered and cesarean placentas [32].

Another limitation of our work is that we did not collect reference material for microbiome analysis from other parts of the body of pregnant women, such as saliva, vaginal secretions or feces. Our main goal was to determine the occurrence of individual bacteria traces, not their origin. However, it is known that the placental core microbiome shares phylotypes with the maternal oral and gut microbiome [32].
