Browsing by Author "Greenberg, Jonathan M."

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    Host-microbiome interactions in distinct subsets of preterm labor and birth
    (2023) Galaz, Jose; Romero, Roberto; Greenberg, Jonathan M.; Theis, Kevin R.; Arenas-Hernandez, Marcia; Xu, Yi; Farias-Jofre, Marcelo; Miller, Derek; Kanninen, Tomi; Garcia-Flores, Valeria; Gomez-Lopez, Nardhy
    Preterm birth, the leading cause of perinatal morbidity, often follows premature labor, a syndrome whose prevention remains a challenge. To better understand the relationship between premature labor and host-microbiome interactions, we conducted a mechanistic investigation using three preterm birth models. We report that intra-amniotic delivery of LPS triggers inflammatory responses in the amniotic cavity and cervico-vaginal microenvironment, causing vaginal microbiome changes and signs of active labor. Intra-amniotic IL-1 alpha delivery causes a moderate inflammatory response in the amniotic cavity but increasing inflammation in the cervico-vaginal space, leading to vaginal microbiome disruption and signs of active labor. Conversely, progesterone action blockade by RU-486 triggers local immune responses accompanying signs of active labor without altering the vaginal microbiome. Preterm labor facilitates ascension of cervico-vaginal bacteria into the amniotic cavity, regardless of stimulus. This study provides compelling mechanistic insights into the dynamic host-microbiome interactions within the cervico-vaginal microenvironment that accompany premature labor and birth.
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    Is there a placental microbiota? A critical review and re-analysis of published placental microbiota datasets
    (2023) Panzer, Jonathan J.; Romero, Roberto; Greenberg, Jonathan M.; Winters, Andrew D.; Galaz, Jose; Gomez-Lopez, Nardhy; Theis, Kevin R.
    The existence of a placental microbiota is debated. The human placenta has historically been considered sterile and microbial colonization was associated with adverse pregnancy outcomes. Yet, recent DNA sequencing investigations reported a microbiota in typical human term placentas. However, this detected microbiota could represent background DNA or delivery-associated contamination. Using fifteen publicly available 16S rRNA gene datasets, existing data were uniformly re-analyzed with DADA2 to maximize comparability. While Amplicon Sequence Variants (ASVs) identified as Lactobacillus, a typical vaginal bacterium, were highly abundant and prevalent across studies, this prevalence disappeared after applying likely  DNA contaminant removal to placentas from term cesarean deliveries. A six-study sub-analysis targeting the 16S rRNA gene V4 hypervariable region demonstrated that bacterial profiles of placental samples and technical controls share principal bacterial ASVs and that placental samples clustered primarily by study origin and mode of delivery. Contemporary DNA-based evidence does not support the existence of a placental microbiota. Importance Early-gestational microbial influences on human development are unclear. By applying DNA sequencing technologies to placental tissue, bacterial DNA signals were observed, leading some to conclude that a live bacterial placental microbiome exists in typical term pregnancy. However, the low-biomass nature of the proposed microbiome and high sensitivity of current DNA sequencing technologies indicate that the signal may alternatively derive from environmental or delivery-associated bacterial DNA contamination. Here we address these alternatives with a re-analysis of 16S rRNA gene sequencing data from 15 publicly available placental datasets. After identical DADA2 pipeline processing of the raw data, subanalyses were performed to control for mode of delivery and environmental DNA contamination. Both environment and mode of delivery profoundly influenced the bacterial DNA signal from term-delivered placentas. Aside from these contamination-associated signals, consistency was lacking across studies. Thus, placentas delivered at term are unlikely to be the original source of observed bacterial DNA signals.
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    Microbiota of the Pregnant Mouse: Characterization of the Bacterial Communities in the Oral Cavity, Lung, Intestine, and Vagina through Culture and DNA Sequencing
    (2022) Greenberg, Jonathan M.; Romero, Roberto; Winters, Andrew D.; Galaz, Jose; Garcia-Flores, Valeria; Arenas-Hernandez, Marcia; Panzer, Jonathan; Shaffer, Zachary; Kracht, David J.; Gomez-Lopez, Nardhy; Theis, Kevin R.
    Mice are frequently used as animal models for mechanistic studies of infection and obstetrical disease, yet characterization of the murine microbiota during pregnancy is lacking. The objective of this study was to characterize the microbiotas of distinct body sites of the pregnant mouse-vagina, oral cavity, intestine, and lung-that harbor microorganisms that could potentially invade the murine amniotic cavity, thus leading to adverse pregnancy outcomes. The microbiotas of these body sites were characterized through anoxic, hypoxic, and oxic culture as well as through 16S rRNA gene sequencing. With the exception of the vagina, the cultured microbiotas of each body site varied by atmosphere, with the greatest diversity in the cultured microbiota appearing under anoxic conditions. Only cultures of the vagina were comprehensively representative of the microbiota observed through direct DNA sequencing of body site samples, primarily due to the predominance of two Rodentibacter strains. Identified as Rodentibacter pneumotropicus and Rodentibacter heylii, these isolates exhibited predominance patterns similar to those of Lactobacillus crispatus and Lactobacillus iners in the human vagina. Whole-genome sequencing of these Rodentibacter strains revealed shared genomic features, including the ability to degrade glycogen, an abundant polysaccharide in the vagina. In summary, we report body site-specific microbiotas in the pregnant mouse with potential ecological parallels to those of humans. Importantly, our findings indicate that the vaginal microbiotas of pregnant mice can be readily cultured, suggesting that mock vaginal microbiotas can be tractably generated and maintained for experimental manipulation in future mechanistic studies of host vaginal-microbiome interactions.
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    No Consistent Evidence for Microbiota in Murine Placental and Fetal Tissues
    (2020) Theis, Kevin R.; Romero, Roberto; Greenberg, Jonathan M.; Winters, Andrew D.; Garcia-Flores, Valeria; Motomura, Kenichiro; Ahmad, Madison M.; Galaz, Jose; Arenas-Hernandez, Marcia; Gomez-Lopez, Nardhy
    The existence of a placental microbiota and in utero colonization of the fetus have been the subjects of recent debate. The objective of this study was to determine whether the placental and fetal tissues of mice harbor bacterial communities. Bacterial profiles of the placenta and fetal brain, lung, liver, and intestine samples were characterized through culture, quantitative real-time PCR (qPCR), and 16S rRNA gene sequencing. These profiles were compared to those of the maternal mouth, lung, liver, uterus, cervix, vagina, and intestine, as well as to background technical controls. Positive bacterial cultures from placental and fetal tissue samples were rare; of the 165 total bacterial cultures of placental tissue samples from the 11 mice included in this study, only nine yielded at least a single colony, and five of those nine positive cultures came from a single mouse. Cultures of fetal intestinal tissue samples yielded just a single bacterial isolate, Staphylococcus hominis, a common skin bacterium. Bacterial loads of placental and fetal brain, lung, liver, and intestinal tissues were not higher than those of DNA contamination controls and did not yield substantive 16S rRNA gene sequencing libraries. From all placental or fetal tissue samples (n = 51), there was only a single bacterial isolate that came from a fetal brain sample having a bacterial load higher than that of contamination controls and that was identified in sequence-based surveys of at least one of its corresponding maternal samples. Therefore, using multiple modes of microbiological inquiry, there was not consistent evidence of bacterial communities in the placental and fetal tissues of mice.
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    The Vaginal Microbiota of Pregnant Women Varies with Gestational Age, Maternal Age, and Parity
    (2023) Romero, Roberto; Theis, Kevin R.; Gomez-Lopez, Nardhy; Winters, Andrew D.; Panzer, Jonathan J.; Lin, Huang; Galaz, Jose; Greenberg, Jonathan M.; Shaffer, Zachary; Kracht, David J.; Chaiworapongsa, Tinnakorn; Jung, Eunjung; Gotsch, Francesca; Ravel, Jacques; Peddada, Shyamal D.; Tarca, Adi L.
    There is debate regarding links between the vaginal microbiota and pregnancy complications, especially spontaneous preterm birth. Inconsistencies in results among studies are likely due to differences in sample sizes and cohort ethnicity.
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    Thymic stromal lymphopoietin participates in the host response to intra-amniotic inflammation leading to preterm labor and birth
    (2023) Kanninen, Tomi; Tao, Li; Romero, Roberto; Xu, Yi; -Hernandez, Marcia Arenas; Galaz, Jose; Liu, Zhenjie; Miller, Derek; Levenson, Dustyn; Greenberg, Jonathan M.; Panzer, Jonathan; Padron, Justin; Theis, Kevin R.; Gomez-Lopez, Nardhy
    The aim of this study was to establish the role of thymic stromal lymphopoietin (TSLP) in the intra-amniotic host response of women with spontaneous preterm labor (sPTL) and birth. Amniotic fluid and chorioamniotic membranes (CAM) were collected from women with sPTL who delivered at term (n = 30) or preterm without intra-amniotic inflammation (n = 34), with sterile intra-amniotic inflammation (SIAI, n = 27), or with intra-amniotic infection (IAI, n = 17). Amnion epithelial cells (AEC), Ureaplasma parvum, and Sneathia spp. were also utilized. The expression of TSLP, TSLPR, and IL-7R & alpha; was evaluated in amniotic fluid or CAM by RT-qPCR and/or immunoassays. AEC co-cultured with Ureaplasma parvum or Sneathia spp. were evaluated for TSLP expression by immunofluorescence and/or RT-qPCR. Our data show that TSLP was elevated in amniotic fluid of women with SIAI or IAI and expressed by the CAM. TSLPR and IL-7R & alpha; had detectable gene and protein expression in the CAM; yet, CRLF2 was specifically elevated with IAI. While TSLP localized to all layers of the CAM and increased with SIAI or IAI, TSLPR and IL-7R & alpha; were minimal and became most apparent with IAI. Co -culture experiments indicated that Ureaplasma parvum and Sneathia spp. differentially upregulated TSLP expression in AEC. Together, these findings indicate that TSLP is a central component of the intra-amniotic host response during sPTL.