*Article* **Comparative Analysis of the Placental Microbiome in Pregnancies with Late Fetal Growth Restriction versus Physiological Pregnancies**

**Aleksandra Stupak 1,\*, Tomasz G ˛eca 1, Anna Kwa´sniewska 1, Radosław Mlak 2, Paweł Piwowarczyk 3, Robert Nawrot 4, Anna Go ´zdzicka-Józefiak <sup>4</sup> and Wojciech Kwa´sniewski <sup>5</sup>**


**Abstract:** A comparative analysis of the placental microbiome in pregnancies with late fetal growth restriction (FGR) was performed with normal pregnancies to assess the impact of bacteria on placental development and function. The presence of microorganisms in the placenta, amniotic fluid, fetal membranes and umbilical cord blood throughout pregnancy disproves the theory of the "sterile uterus". FGR occurs when the fetus is unable to follow a biophysically determined growth path. Bacterial infections have been linked to maternal overproduction of pro-inflammatory cytokines, as well as various short- and long-term problems. Proteomics and bioinformatics studies of placental biomass allowed the development of new diagnostic options. In this study, the microbiome of normal and FGR placentas was analyzed by LC-ESI-MS/MS mass spectrometry, and the bacteria present in both placentas were identified by analysis of a set of bacterial proteins. Thirty-six pregnant Caucasian women participated in the study, including 18 women with normal pregnancy and eutrophic fetuses (EFW > 10th percentile) and 18 women with late FGR diagnosed after 32 weeks of gestation. Based on the analysis of the proteinogram, 166 bacterial proteins were detected in the material taken from the placentas in the study group. Of these, 21 proteins had an exponentially modified protein abundance index (emPAI) value of 0 and were not included in further analysis. Of the remaining 145 proteins, 52 were also present in the material from the control group. The remaining 93 proteins were present only in the material collected from the study group. Based on the proteinogram analysis, 732 bacterial proteins were detected in the material taken from the control group. Of these, 104 proteins had an emPAI value of 0 and were not included in further analysis. Of the remaining 628 proteins, 52 were also present in the material from the study group. The remaining 576 proteins were present only in the material taken from the control group. In both groups, we considered the result of ns prot ≥ 60 as the cut-off value for the agreement of the detected protein with its theoretical counterpart. Our study found significantly higher emPAI values of proteins representative of the following bacteria: *Actinopolyspora erythraea*, *Listeria costaricensis*, *E. coli*, *Methylobacterium*, *Acidobacteria bacterium*, *Bacteroidetes bacterium*, *Paenisporsarcina* sp., *Thiodiazotropha endol oripes* 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*. Our study showed that placental dysbiosis may be an important factor in the etiology of FGR. The presence of numerous bacterial proteins present in the control material may indicate their protective role, while the presence of bacterial proteins detected only in the material taken from the placentas of the study group may indicate their potentially pathogenic nature. This phenomenon is probably important in the development of the immune system in early life, and the placental microbiota and its metabolites may have great potential in the screening, prevention, diagnosis and treatment of FGR.

**Citation:** Stupak, A.; G ˛eca, T.; Kwa´sniewska, A.; Mlak, R.; Piwowarczyk, P.; Nawrot, R.; Go ´zdzicka-Józefiak, A.; Kwa´sniewski, W. Comparative Analysis of the Placental Microbiome in Pregnancies with Late Fetal Growth Restriction versus Physiological Pregnancies. *Int. J. Mol. Sci.* **2023**, *24*, 6922. https:// doi.org/10.3390/ijms24086922

Academic Editor: Maria Teresa Mascellino

Received: 6 March 2023 Revised: 3 April 2023 Accepted: 6 April 2023 Published: 7 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** microbiome; bacteria; proteome; pregnancy; FGR; placenta
