Walther-António, Marina

Giving birth too soon accounts for half of all newborn deaths worldwide. Clinical symptoms alone are not sufficient to identify women at risk of giving birth too early, as such a pragmatic approach to reducing the incidence of preterm birth entails developing early strategies for intervention before it materializes.

Abstract Incidence rates for endometrial cancer (EC) are rising, particularly in postmenopausal and obese women. Our previous work has showed that the uterine and vaginal microbiome distinguishes patients with EC from those without. We now examine the impact of patient factors (such as menopause status, body mass index, and vaginal pH) in the microbiome in the absence of EC and how these might contribute to the microbiome signature in EC. We find that each factor independently alters the microbiome and identified postmenopausal status as the main driver of a polymicrobial network associated with EC (ECbiome). Of the 17 taxa we found enriched in EC patients, 8 were also enriched by postmenopause. Because postmenopausal status is a main risk factor for endometrial cancer, this system can be thought of as an ecological succession towards a disease state. Organisms within the ECbiome have been previously identified as polymicrobial associations in chronic soft tissue infections, ulcers, morbid obesity, and bacterial vaginosis. This suggests that this microbiome network is likely involved in mechanisms of host disease. Within the ECbiome, Porphyromonas somerae stands out because of its very significant and specific association with EC (p = 5.73E-6; Q = 0.009). We had previously identified this species as significantly associated with EC, and hereby validate that finding. Because the association of P. somerae with EC was robust and specific enough to potentially be considered as a biomarker for the disease (AUC 76.7%, CI: 67.9-85.5%), we developed a P. somerae-specific qPCR assay to detect this species in vaginal swabs. This becomes of particular interest because the lower tract and uterine microbiome are significantly correlated in these patients (p = 0.01), indicating that the vaginal microbiome can be used as a proxy for the uterine environment in EC. In agreement with the 16S rRNA gene sequencing data, P. somerae was detected significantly more frequently in vaginal swabs of patients with EC than those without EC (p = 3.02E-7). Overall, the test achieved a sensitivity of 74% and specificity of 63%, with a positive predictive value of 0.86 for obese and postmenopausal patients. In summary, we identify Porphyromas somerae presence as the most predictive microbial marker of EC, and we confirm this using targeted qPCR, which could be of use in detecting EC in high-risk, asymptomatic women. Given the established pathogenic behavior of P.somerae, we have also explored mechanistic routes through which this microbe and associated consortia may be involved in the mechanisms of disease. Citation Format: Marina Walther-Antonio, Dana Walsh, Alexis Hokenstad, Jun Chen, Jayeun Sung, Gregory Jenkins, Nicholas Chia, Heidi Nelson, Andrea Mariani. Postmenopause as a key factor in the composition of the Endometrial Cancer Microbiome (ECbiome): Putative role of Porphyromonas somerae in the disease [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR09.

The vaginal microbiome is a critical component of women’s sexual and reproductive health, with variations in microbial composition, particularly the loss of Lactobacillus species, being implicated in gynecologic and obstetric diseases. Given that the vaginal microbiome is so crucial, why do vaginal microbial profiles vary strikingly from person to person and even change over time within the same person? In the present study, which tracked the daily vaginal microbiomes of young healthy women through different lifestyles, we found that use of a locally released progestin contraceptive, a vegetarian diet, and intense exercise appear to lead to vaginal microbiome alterations and loss of Lactobacillus species. The impact of these vaginal microbiome changes on immediate and long-term health remain to be investigated.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory, cholestatic liver disease of unknown etiopathogenesis which is recognized as a major risk factor for cholangiocarcinoma (CCA). We aimed to investigate the roles of the biliary and intestinal microflora in the inflammatory process of PSC and to determine if the microflora is associated with progression to CCA. METHODS: We studied the bile and stool microbiomes in samples obtained from patients with PSC (31 stool and 35 bile samples), sporadic CCA (11 stool and 26 bile samples), and PSC coexisting with CCA (16 stool and bile 17 samples). Control bile was obtained from patients with cholelithiasis or choledocholithiasis (n = 17). DNA extraction was followed by amplification and high-throughput next generation sequencing (MiSeq) of the 16S rDNA (V3-V5) region to identify the microbiota present. We performed: 1. analysis of the stool microbiome between PSC and CCA patients; 2. analysis of the biliary microbiome in PSC and CCA patients; 3. comparison of the biliary microbiome collected before and after biliary stent placement. RESULTS: Although bile samples from study patients had significantly lower sequencing depths than stool, their sequencing depths were much higher than bile from negative controls. The microbiomes of stool and bile samples from the same patients were significantly correlated ( P = 0.022). CCA bile samples had significant difference of alpha diversity from control bile samples with less Streptococcaceae and Desulfovibrionaceae (FDR < 10%) than PSC whereas no significant change of stool microbiome was identified between CCA and PSC. Fusobacterium and Gemella were correlated to PSC duration. An increased abundance of Fusobacteria after stent placement was revealed. Fusobacteria, which is inflammation-promoting, was also increased with the number of stents in the bile duct. CONCLUSION: Our study suggests that the bile microbiome is potentially a more distinctive biomarker than the stool microbiome for PSC and CCA development. There is increased inhabitance of inflammation-promoting bacteria in the bile of CCA patients and after biliary stent placement. Increased PSC duration also increases the microbial burden in the bile, which may also promote CCA development. We further detected specific bile microbes statistically associated with CCA or stent presence in the bile ducts, which raises the possibility of their role in the CCA progression or stent obstruction. Additional analyses with more samples are needed.

Single cell sequencing is a technology capable of analyzing the genome of a single cell within a population. This technology is mostly integrated with microfluidics for precise cell manipulation and fluid handling. So far, most of the microfluidic-based single cell genomic studies have been focused on lab-cultured species or cell lines that are relatively easy to handle following standard microfluidic-based protocols without additional adjustments. The major challenges for performing single cell sequencing on clinical samples is the complex nature of the samples which requires additional sample processing steps to obtain intact single cells of interest without using amplification-inhibitive agents. Fluorescent-activated cell sorting is a common option to obtain single cells from clinical samples for single cell applications but requires &gt;100 000 viable cells in suspension and the need for specialized laboratory and personnel. In this work, we present a protocol that can be used to obtain intact epithelial cells from snap-frozen postsurgical human endometrial tissues for single cell whole genome amplification. Our protocol includes sample thawing, cell dissociation, and labeling for genome amplification of targeted cells. Between 80% and 100% of single cell replicates lead to &gt;25 ng of DNA after amplification with no measurable contamination, sufficient for downstream sequencing.

The microbe-stuffed gut, rather than the genome, represents the most dynamic gene reservoir within complex, multicellular metazoa (animals). Microbes are known to confer increased metabolic efficiency, increased nutrient recovery, and tolerance of ocean acidity to basal taxa such as sponges, arguably the extant taxa most comparable to the first metazoan. We hypothesize that metazoan origins may be rooted in the capability to compartmentalize, metabolize, and exchange genetic material with a modulated microbiome. We present evidence that the most parsimonious adaptive response of clonal eukaryotic colonies experiencing oligotrophic (nutrient-limited) conditions that accompanied Neoproterozoic glaciation events, which were broadly contemporaneous with metazoan origins, is to evolve a morphological volume to harbor a densified microbiome. Dense microbial communities housed within a cavity would increase instances of horizontal gene transfer between microorganisms and host, accelerating evolutionary innovation at the genetic and epigenetic levels for the holobiont. The accelerated tempo of genetic exchange would continue until the host’s metabolic and reproductive cells became spatially and temporally segregated from one another, at which point the process is effectively suppressed with the emergence of specialized gut and reproductive tissues. This framework may lead to new, testable hypotheses regarding metazoan evolution on Earth and a more tractable means of estimating the pervasiveness of complex, multicellular animal-like life with convergent morphologies on other planets.

Abstract Ovarian cancer (OC) is the most lethal gynecologic malignancy and the fifth leading cause of cancer death in the United States. Metformin, an antidiabetic drug, has been shown to decrease the risk of mortality in cancer patients, particularly those with OC. Though the drug acts through inhibition of the PI3K/mTOR/AKT pathway, which is commonly altered in OC, knockout of key proteins in the pathway does not attenuate metformin’s antidiabetic effects. Further, intravenous metformin administration is less effective than oral, implying that some crucial interaction takes place in the gut. The gut is home to some 1013 bacterial cells, collectively known as the microbiota. These bacteria play important roles in fermentation of fiber, producing short-chain fatty acids (SCFA) that have positive health benefits, including prevention of metabolic syndrome, bowel disorders, and certain cancers. Mechanistically, SCFAs inhibit the PI3K/mTOR/AKT pathway, similar to metformin. Conditions such as obesity, metabolic syndrome, and diabetes significantly alter gut microbiota composition, possibly leading to a reduction in beneficial SCFAs. However, in type II diabetes, treatment with metformin leads to an increase in SCFA-producing bacteria, suggesting that metformin may positively influence the microbiota to improve patient metabolic profiles. In OC, metformin may play a similar role in promoting the presence of SCFA-producing bacteria. We hypothesized that metformin increases the abundance of SCFA-producing bacteria in the gut, with which it acts synergistically to decrease growth of OC cells in combination with OC chemotherapy. To investigate our hypothesis, we have employed the OC patient-derived xenograft (PDX) mouse model established at Mayo Clinic. Immunocompromised SCID mice received OC cells from Mayo Clinic patients and the tumors were allowed to grow to an appropriate size before treatment was started. Cage mates were split into one of four treatment arms: a control (no treatment) group, metformin only, carboplatin/paclitaxel (CP) chemotherapy only, or CP with metformin. Mice received 2mg/ml metformin in their water continuously and/or 51mg/kg carboplatin and 15mg/kg paclitaxel intravenously weekly. Stool was collected weekly prior to tumor injection, during tumor growth, and through the four weeks of treatment. DNA was extracted from stool and bacterial 16S rRNA amplicon sequencing was used to determine microbiota profiles. Mice with chemosensitive tumors treated with only CP chemotherapy had significant reductions in tumor growth along with significantly less microbial gut diversity than pretumor and pretreatment mice. Chemosensitive mice treated with metformin alone did not lose significant microbial diversity but did not have significant reductions in tumor growth as compared to the control mice. CP in combination with metformin both reduced tumor growth and protected against loss of microbiota diversity. Further, mice treated with only metformin had significant increases in abundance in the Firmicutes genera Ruminococcus and Oscillospira, SCFA producers that are associated with leanness and health. Mice treated with both metformin and CP had significant increases in another SCFA-producing Firmicute, Moryella. Finally, mice with chemoresistant tumors showed improved tumor growth inhibition with combination CP/metformin therapy, suggesting that metformin-associated changes in the gut microbiota may increase sensitivity to CP. These data support the hypothesis that metformin protects against detrimental loss of gut microbiota diversity induced by chemotherapy treatment, enriching beneficial SCFA producers. This work implies a role for the gut microbiota in OC treatment and provides support for metformin’s potential use as a beneficial addition to chemotherapy treatment. Citation Format: Dana M. Walsh, Ismail Mert, Chen Jun, Xiaonan Hou, Saravut John Weroha, Marina Walther-Antonio. Metformin alters the gut microbiota of ovarian cancer patients treated with carboplatin/paclitaxel chemotherapy and enhances sensitivity in resistant tumors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A70.

Abstract In this review, we aimed to provide insight into the microbiome and its association with endometrial and ovarian cancer and their risk factors. We reviewed the literature focusing on the relationship between the microbiome and cancer, as well as the relationship between gynecologic diseases and cancers. The human body contains different kinds of microorganisms in various body parts, which is termed the microbiome . The number of microorganisms that live in and on the human body is greater than that of the human germ and somatic cells by 10‐fold. The relationship between a human and their microbiome is complex; it is also one of the most important components of homeostasis. Impairment of microbiome–host homeostasis has been associated with obesity, several cancers, preterm labor, inflammatory and allergic conditions and neurodevelopmental disorders. Direct and strong causal relationships have been established for several cancers and microorganisms, such as gastric lymphoma and Helicobacter pylori infection. Interestingly, eradication of the infectious agents has also been shown to be therapeutic. The association between cancer and the microbiome, however, is more complicated than a 1 bacteria–1 cancer model, and a shift in a healthy microbiome can result in various cancers via inflammation, change in microenvironment or DNA‐damaging toxins. The human microbiome is an integral part of homeostasis. Understanding the mechanisms that cause dysbiosis will enable us to elucidate the pathways that result in malignancy and investigate new treatment modalities.

Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.