Lack of evidence for microbiota in the placental and fetal tissues of rhesus macaques

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The prevailing paradigm in obstetrics has been the sterile womb hypothesis. However, some are asserting that the placenta, intra-amniotic environment, and fetus harbor microbial communities. The objective of this study was to determine if the fetal and placental tissues of rhesus macaques harbor viable bacterial communities. Fetal, placental, and uterine wall samples were obtained from cesarean deliveries without labor (∼130/166 days gestation). The presence of viable bacteria in the fetal intestine and placenta was investigated through culture. The bacterial burden and profile of the placenta, umbilical cord, and fetal brain, heart, liver, and colon were determined through quantitative real-time PCR and DNA sequencing. These data were compared with those of the uterine wall, as well as to negative and positive technical controls. Bacterial cultures of fetal and placental tissues yielded only a single colony of <jats:italic>Cutibacterium acnes</jats:italic>. This bacterium was detected at a low relative abundance (0.02%) in the 16S rRNA gene profile of the villous tree sample from which it was cultured, yet it was also identified in 12/29 background technical controls. The bacterial burden and profile of fetal and placental tissues did not exceed or differ from those of background technical controls. In contrast, the bacterial burden and profiles of positive controls exceeded and differed from those of background controls. Among the macaque samples, distinct microbial signals were limited to the uterine wall. Therefore, using multiple modes of microbiologic inquiry, there was not consistent evidence of viable bacterial communities in the fetal and placental tissues of rhesus macaques.</jats:p><jats:sec><jats:title>IMPORTANCE</jats:title><jats:p>Microbial invasion of the amniotic cavity (i.e. intra-amniotic infection) has been causally linked to pregnancy complications, especially preterm birth. Therefore, if the placenta and the fetus are typically populated by low biomass yet viable microbial communities, current understanding of the role of microbes in reproduction and pregnancy outcomes will need to be fundamentally reconsidered. Could these communities be of benefit by competitively excluding potential pathogens or priming the fetal immune system for the microbial bombardment it will experience upon delivery? If so, what properties (e.g. microbial load, community membership) of these microbial communities preclude versus promote intra-amniotic infection? Given the ramifications of the <jats:italic>in utero</jats:italic> colonization hypothesis, critical evaluation is required. In this study, using multiple modes of microbiologic inquiry (i.e. culture, qPCR, DNA sequencing) and controlling for potential background DNA contamination, we did not find consistent evidence for microbial communities in the placenta and fetal tissues of rhesus macaques.</jats:p></jats:sec>

Maternal-Fetal Immune Responses in Pregnant Women Infected with SARS-CoV-2

“…Traditionally, the placenta is considered a sterile organ 107 , 108 . Indeed, recent research has reiterated the sterile womb hypothesis using placentas from women who delivered via cesarean section at term without labor 109 – 111 as well as studies in mice 112 , 113 and non-human primates 114 . Here, we evaluated the possibility that maternal SARS-CoV-2 infection compromises the sterility of the placenta by facilitating the invasion of bacteria or the transfer of bacterial DNA from maternal compartments.…”

“…All DNA extractions were completed within a biological safety cabinet using a DNeasy Powerlyzer Powersoil Kit (Qiagen, Germantown, MD, USA), with minor modifications to the manufacturer’s instructions as previously described 112 , 114 . Personnel wore sterile surgical masks, gowns, and gloves during the procedure.…”

“…Total bacterial DNA abundance within samples was measured via amplification of the V1 - V2 region of the 16S rRNA gene according to the protocol of Dickson et al 140 with minor modifications, as previously described 112 , 114 . These modifications included the use of a degenerative forward primer (27f-CM: 5’-AGA GTT TGA TCM TGG CTC AG-3’) and a degenerate probe containing locked nucleic acids (+) (BSR65/17: 5’−56FAM-TAA +YA+C ATG +CA+A GT+C GA-BHQ1–3’).…”

“…16S rRNA gene sequences were clustered into amplicon sequence variants (ASVs) defined by 100% sequence similarity using DADA2 version 1.12 142 in R version 3.6.1 143 according to the online MiSeq protocol ( https://benjjneb.github.io/dada2/tutorial.html ) with minor modifications, as previously described 114 . These modifications included allowing truncation lengths of 250 and 150 bases, and a maximum number of expected errors of 2 and 7 bases, for forward and reverse reads, respectively.…”

Bioinformatics Analysis of Oral, Vaginal, and Rectal Microbial Profiles during Pregnancy: A Pilot Study on the Bacterial Co-Residence in Pregnant Women

“…However, simple vaginal bacterial profiling does not fully elucidate the mechanism of how vaginal microbes could cause or prevent TPL and PTB or BV-associated diseases, since other commensal microbes, such as oral or gut microbes, could influence pregnancy outcomes [ 2 , 23 ]. Indeed, a similarity of profiles has been observed between the oral and placental microbiota [ 24 ], raising an issue of periodontal microbes influencing the maintenance of pregnancy [ 2 , 25 , 26 , 27 ], although several recent studies claim that the placenta is indeed sterile [ 28 , 29 , 30 ]. In addition, the association of intestinal microbiota with BV determined by PCR analysis has also been reported [ 31 , 32 ], suggesting that the microbiome in other locations could be associated with CST IV, where BV-associated TPL and PTB are evident [ 19 ].…”