Potential tools for predicting response to chemotherapy in OC: Assessment of immune dysbiosis, participant's self-rated health and microbial dynamics.

Epithelial ovarian cancer (OC) is the deadliest female reproductive cancer; an estimated 13,270 women will die from OC in 2023. Platinum-based chemotherapy resistance mechanisms contribute to poor OC 5-year survival rates. Peripheral inflammation is linked to various disease states and we previously identified unique peritoneal microbial features predictive of OC. We hypothesized that unique peripheral immune profiles and peritoneal microbial features may be predictive of disease-free interval (time to recurrence) and response to chemotherapy in participants with OC. We also investigated self-rated health (SRH) scores in the context of peripheral inflammation as a potential screening tool for OC. Blood and peritoneal fluid were collected from participants with OC or a benign adnexal mass (BPM). Lymphocyte populations were analyzed using Fluorescence Activated Cell Sorting, serum cytokine levels were analyzed using the Human Th17 Magnetic Bead Panel assay and peritoneal fluid microbial features were analyzed using Next Generation Sequencing (NGS). Participants completed a standardized questionnaire on self-rated physical and emotional health. Participants were classified into three chemotherapy response categories: platinum-refractory, platinum-resistant or platinum-sensitive. A significant positive correlation was found between elevated inflammatory status on the day of surgery and longer disease-free interval. SRH measures did not correlate with immune status in participants with OC or a BPM. We identified a correlation between peritoneal microbial features and chemotherapy response. We conclude that immune dysbiosis may be useful in predicting OC recurrence. The immune findings reported here set the framework for additional studies utilizing immune profiles to predict platinum-based chemotherapy responsiveness in OC.

Evaluation of sterol­o­acyl transferase 1 and cholesterol ester levels in plasma, peritoneal fluid and tumor tissue of patients with endometrial cancer: A pilot study.

Endometrial cancer (EC) is the most prevalent gynecological malignancy. Abnormal accumulation of sterol-O-acyl transferase 1 (SOAT1) and SOAT1-mediated cholesterol ester (CE) contributes to cancer progression in various malignancies, including ovarian cancer. Therefore, it was hypothesized that similar molecular changes may occur in EC. The present study aimed to evaluate the diagnostic and/or prognostic potential of SOAT1 and CE in EC by: i) Determining SOAT1 and CE levels in plasma, peritoneal fluid and endometrial tissue from patients with EC and control subjects; ii) performing receiver operating characteristic curve analysis to determine diagnostic performance; iii) comparing SOAT1 and CE expression to that of the tumor proliferation marker Ki67; and iv) assessing the association between SOAT1 expression and survival. Enzyme-linked immunosorbent assay was used to determine the levels of SOAT1 protein in tissue, plasma and peritoneal fluid. The mRNA and protein expression levels of SOAT1 and Ki67 in tissues were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. CE levels were determined colorimetrically in plasma and peritoneal fluid. SOAT1-associated survival data from the cBioPortal cancer genomics database were used to assess prognostic relevance. The results revealed that SOAT1 and CE levels were significantly elevated in tumor tissue and peritoneal fluid samples collected from the EC group. By contrast, the plasma levels of SOAT1 and CE in the EC and control groups were similar. Significant positive associations between CE and SOAT1, SOAT1/CE and Ki67, and SOAT1/CE and poor overall survival in patients with EC suggested that SOAT1/CE may be associated with malignancy, aggressiveness and poor prognosis. In conclusion, SOAT1 and CE may serve as potential biomarkers for prognosis and target-specific treatment of EC.

Effects of endometriosis on immunity and mucosal microbial community dynamics in female olive baboons.

Endometriosis is defined as the growth of endometrial tissue in ectopic locations, and is associated with altered immune and microbial phenotypes. It is unclear if these changes are the result of the disease or may be causative. We induced endometriosis in non-human primates (Papio Anubis) to test our hypothesis that the growth of endometriotic lesions results in alterations in immune and microbial dynamics that may advance disease progression. Baboon samples were collected pre-inoculation (prior to disease induction), at 3, 6, 9, and 15 months after disease induction. Tolerant regulatory T-cells (Tregs) and inflammatory T-helper 17 (Th17) cells were identified in peripheral blood and within the eutopic/ectopic endometrial tissues. Microbiome communities were identified in fecal/urine samples. The induction of endometriosis decreased peripheral Tregs cells while Th17 cells increased at all post-induction collections, thus reducing the Tregs:Th17 cells ratio, indicating systemic inflammation. Microbiome diversity and abundance were altered at each sample site after disease induction. Thus, induction of endometriosis in baboons caused an immune shift toward an inflammatory profile and altered mucosal microbial profiles, which may drive inflammation through production of inflammatory mediators. Immune and microbial profiling may lead to innovative diagnostic tools and novel therapies for endometriosis treatment.

Association of microbial dynamics with urinary estrogens and estrogen metabolites in patients with endometriosis.

Endometriosis is an estrogen dependent gynecological disease associated with altered microbial phenotypes. The association among endogenous estrogen, estrogen metabolites, and microbial dynamics on disease pathogenesis has not been fully investigated. Here, we identified estrogen metabolites as well as microbial phenotypes in non-diseased patients (n = 9) and those with pathologically confirmed endometriosis (P-EOSIS, n = 20), on day of surgery (DOS) and ~1-3 weeks post-surgical intervention (PSI). Then, we examined the effects of surgical intervention with or without hormonal therapy (OCPs) on estrogen and microbial profiles of both study groups. For estrogen metabolism analysis, liquid chromatography/tandem mass spectrometry was used to quantify urinary estrogens. The microbiome data assessment was performed with Next generation sequencing to V4 region of 16S rRNA. Surgical intervention and hormonal therapy altered gastrointestinal (GI), urogenital (UG) microbiomes, urinary estrogen and estrogen metabolite levels in P-EOSIS. At DOS, 17ß-estradiol was enhanced in P-EOSIS treated with OCPs. At PSI, 16-keto-17ß-estradiol was increased in P-EOSIS not receiving OCPs while 2-hydroxyestradiol and 2-hydroxyestrone were decreased in P-EOSIS receiving OCPs. GI bacterial α-diversity was greater for controls and P-EOSIS that did not receive OCPs. P-EOSIS not utilizing OCPs exhibited a decrease in UG bacterial α-diversity and differences in dominant taxa, while P-EOSIS utilizing OCPs had an increase in UG bacterial α-diversity. P-EOSIS had a strong positive correlation between the GI/UG bacteria species and the concentrations of urinary estrogen and its metabolites. These results indicate an association between microbial dysbiosis and altered urinary estrogens in P-EOSIS, which may impact disease progression.

Altered eutopic endometrial T-regulatory and T-helper 17 lymphocyte ratio in women with unexplained subfertility.

PROBLEM: Perturbations in T-helper lymphocyte profiles have previously been associated with endometriosis related subfertility and conception failure. Hence a retrospective in vitro study was conducted to evaluate the relationship between T-regulatory (Treg) and T-helper 17 (Th17) lymphocytes in the eutopic endometrium of women with unexplained subfertility and correlate these profiles to their conception status. METHOD OF STUDY: Eutopic endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle, from women with unexplained subfertility. These samples were evaluated immunohistochemically for Treg and Th17 lymphocytes as well as the related proinflammatory cytokine, Interleukin-17 (IL-17). These eutopic endometrial T lymphocyte subpopulations were compared to the patients' conception status in subsequent cycles. RESULTS: Though Treg cells were not indicative of conception success in subsequent cycles, patients who maintained their subfertile (no conception) status were observed to have a higher Th17 cell count in their eutopic endometrium. The ratio of Treg:Th17 cell counts was significantly correlated to patient conception status as well. These trends stayed consistent irrespective of concurrent endometriosis. CONCLUSION: Patients with a high proinflammatory Th17 lymphocyte profile and low Treg:Th17 ratio in their eutopic endometrium during the secretory phase of their menstrual cycle are more likely to not conceive in subsequent cycles.

Virilizing doses of testosterone decrease circulating insulin levels and differentially regulate insulin signaling in liver and adipose tissue of females.

Transgender men undergoing hormone therapy are at risk for insulin resistance. However, how virilizing testosterone therapy affects serum insulin and peripheral insulin sensitivity in transgender men is unknown. This study assessed the effect of acute, virilizing testosterone on serum insulin concentrations and insulin signaling in liver, skeletal muscle, and white adipose tissue (WAT) of female pigs as a translational model for transgender men. Females received three doses of intramuscular testosterone cypionate (TEST females; 50 mg/day/pig) or corn oil (control) spaced 6 days apart starting on the day of estrus (D0). Fasting blood was collected on D0, D3, D5, D11, and D13, and females were euthanized on D13. On D13, TEST females had virilizing concentrations of serum testosterone with normal concentrations of serum estradiol. Virilizing serum testosterone concentrations (D13) were associated with decreased serum insulin and C-peptide concentrations. Blood glucose and serum glycerol concentrations were not altered by testosterone. Virilizing concentrations of testosterone downregulated AR and ESR1 in subcutaneous (sc) WAT and upregulated transcript levels of insulin-signaling pathway components in WAT and liver. At the protein level, virilizing testosterone concentrations were associated with increased PI3K 110α in liver and increased insulin receptor (INSR) and phospho(Ser256)-FOXO1 in visceral (v) WAT but decreased phospho(Ser473)-AKT in vWAT and scWAT. These results suggest that acute exposure to virilizing concentrations of testosterone suppresses circulating insulin levels and results in increased abundance of proteins in the insulin-signaling pathway in liver and altered phosphorylation of key proteins in control of insulin sensitivity in WAT.NEW & NOTEWORTHY Acute virilizing doses of testosterone administered to females suppress circulating insulin levels, upregulate components of the insulin-signaling pathway in liver, and suppress insulin signaling in white adipose tissue. These results suggest that insulin resistance in transgender men may be due to suppression of the insulin-signaling pathway and decreased insulin sensitivity in white adipose tissue.

Alteration of systemic and uterine endometrial immune populations in patients with endometriosis.

PROBLEM: Endometriosis is defined as growth of endometrial tissue in ectopic locations; it is associated with infertility and chronic pain and affects ~12% of reproductive-aged women. Although inflammation is known to play a key role in endometriosis, knowledge related to immune phenotypes associated with this disease is lacking. This study aimed to characterize immune profiles in patients with endometriosis, to assess inflammatory mediators, to and determine if surgical and/or hormonal therapies restore immune homeostasis. METHODS OF STUDY: Samples from nine controls and 20 histologically confirmed endometriosis patients were collected upon surgery and ~1-3 weeks post-surgical intervention. Subjects were either not utilizing hormonal suppression or were currently on monophasic hormonal therapy. Tolerant regulatory T cells (Tregs = natural [nTregs] +inducible [iTregs]) and inflammatory T helper 17 (Th17) cells were identified in peripheral blood via flow cytometry and within the eutopic/ectopic endometrial tissues via immunohistochemistry and real-time-qPCR. Cytokines were assessed via 10-plex-ELISA. RESULTS: Patients with endometriosis not utilizing hormonal therapy exhibited lower iTregs (tolerant), greater Th17 (inflammatory), and a reduction in Treg/Th17 ratio (P < .05), indicative of systemic inflammation. Treg and Th17 localizations were enhanced within the ectopic endometrial implant, which promotes lesion development. Hormonal therapy decreased systemic and local inflammation (eutopic/ectopic endometrium) via decreased iTregs and Th17 cells in patients with endometriosis (P < .05). Thus, imbalance within immune populations correlated with increased inflammation in patients with endometriosis, which was mitigated by hormonal therapy. CONCLUSIONS: Patients with endometriosis exhibited systemic and localized inflammation within ectopic and endometrial tissues. Hormonal therapy dampened inflammation caused by disease.

Dietary substitution of soybean oil with coconut oil in the absence of dietary antibiotics supports growth performance and immune function in nursery and grower pigs.

BACKGROUND: We hypothesized that supplementation of nursery and grower pig diets with coconut oil in the absence of antibiotics would yield maintenance of glucose homeostasis, growth performance, and immune function similar to what is achieved with nursery and grower pig diets containing antibiotics. Pigs received the same base treatment diets from d24 (weaning) to d71 of age and had blood and fecal samples collected on d24, d31, d45 and d71 for measurement of whole blood glucose, serum insulin, cortisol and cytokines, and fecal microbiome. Pigs had weekly weights and daily feed consumption measured throughout the study. Animals were euthanized at d71 and subcutaneous fat and ileal contents were collected for assessment for fatty acids and microbiome, respectively. Diet treatments consisted of 2% soybean oil plus antibiotics (ABX; n = 22), 2% soybean oil without antibiotics (NABX; n = 22), and 2% coconut oil without antibiotics (COC; n = 22). Statistical analysis examined the effect of diet within each timepoint using a repeated measures ANOVA. RESULTS: Pigs fed COC diet had decreased serum insulin levels, maintained feed intake, feed conversion and weight gain, and, based on serum cytokines and fecal microbiome, were immunologically similar to ABX-fed pigs. However, NABX-fed pigs performed similarly to the ABX-fed pigs in all parameters except for serum cytokines. Additionally, there was no difference in the incidence of diarrhea between any of the diet treatments. CONCLUSIONS: This study demonstrates that dietary antibiotics are not necessary to maintain growth performance in nursery and grower pigs. However, dietary antibiotics appear to modulate circulating cytokine levels. Dietary coconut oil is neither harmful nor helpful to growth performance or immune function in nursery and grower pigs but does modulate serum insulin levels. Therefore, while coconut oil fed at 2% by weight is a suitable substitute for dietary antibiotics, this study suggests that no substitute for dietary antibiotics is needed at all.

Assessment of peritoneal microbial features and tumor marker levels as potential diagnostic tools for ovarian cancer.

Epithelial ovarian cancer (OC) is the most deadly cancer of the female reproductive system. To date, there is no effective screening method for early detection of OC and current diagnostic armamentarium may include sonographic grading of the tumor and analyzing serum levels of tumor markers, Cancer Antigen 125 (CA-125) and Human epididymis protein 4 (HE4). Microorganisms (bacterial, archaeal, and fungal cells) residing in mucosal tissues including the gastrointestinal and urogenital tracts can be altered by different disease states, and these shifts in microbial dynamics may help to diagnose disease states. We hypothesized that the peritoneal microbial environment was altered in patients with OC and that inclusion of selected peritoneal microbial features with current clinical features into prediction analyses will improve detection accuracy of patients with OC. Blood and peritoneal fluid were collected from consented patients that had sonography confirmed adnexal masses and were being seen at SIU School of Medicine Simmons Cancer Institute. Blood was processed and serum HE4 and CA-125 were measured. Peritoneal fluid was collected at the time of surgery and processed for Next Generation Sequencing (NGS) using 16S V4 exon bacterial primers and bioinformatics analyses. We found that patients with OC had a unique peritoneal microbial profile compared to patients with a benign mass. Using ensemble modeling and machine learning pathways, we identified 18 microbial features that were highly specific to OC pathology. Prediction analyses confirmed that inclusion of microbial features with serum tumor marker levels and control features (patient age and BMI) improved diagnostic accuracy compared to currently used models. We conclude that OC pathogenesis alters the peritoneal microbial environment and that these unique microbial features are important for accurate diagnosis of OC. Our study warrants further analyses of the importance of microbial features in regards to oncological diagnostics and possible prognostic and interventional medicine.

The effect of the urinary and faecal microbiota on lower urinary tract symptoms measured by the International Prostate Symptom Score: analysis utilising next-generation sequencing.

OBJECTIVE: To examine the correlation between urinary and faecal microbial profiles and the different aspects of lower urinary tract symptoms (LUTS) in men, as there is accumulating evidence that variations in the human microbiota may promote different benign disease conditions. PATIENTS AND METHODS: We extracted total DNA from urine and faecal samples of a group of men, under an Institutional Review Board-approved protocol. At the same time, International Prostate Symptom Score (IPSS) data were collected. We then amplified the extracted DNA and sequenced it using bacterial 16S ribosomal RNA gene high-throughput next-generation sequencing platform, and analysed the microbial profiles for taxonomy to examine the correlation between the different operational taxonomy units (OTUs) and LUTS represented by the total IPSS, the different symptom levels of the IPSS (mild, moderate, and severe) and its subcomponents of storage, nocturia, voiding, and bother. RESULTS: We included 30 patients (60 samples; one urine and one faecal per patient). In all, 48 faecal OTUs showed a significant correlation with one or more of the IPSS components; 27 with nocturia, 19 with bother, 16 with storage symptoms, and nine with voiding symptoms. The most substantial negative (protective) correlation was between Lachnospiraceae Blautia, a bacteria that increases the availability of gut anxiolytic and antidepressant short-chain fatty acids, and bother (correlation coefficient 0.702; P = 0.001). The abundance of L. Blautia continued to have a protective correlation against LUTS when looking at the different levels of IPSS severity (moderate and severe vs mild, correlation coefficient 0.6132; P = 0.002). Ten unique urinary OTUs showed significant correlation with LUTS; eight with nocturia, one with bother, three with storage, and one with voiding, but no faecal OUT had more than a low correlation with the outcomes of interest in this study. CONCLUSIONS: Our prospective work finds a plausible correlation between L. Blautia and LUTS. Additional studies are needed to determine if the correlations found in the present research are applicable to the general population of patients affected by LUTS.

Prospective examination of the changes in the urinary microbiome induced by transrectal biopsy of the prostate using 16S rRNA gene analysis.

OBJECTIVES: To prospectively examine the changes in microbiota within the urinary tract after transrectal prostate biopsy. MATERIALS AND METHODS: Data, urine, and fecal samples prospectively collected from 30 patients before and after transrectal biopsy of the prostate. DNA was extracted from urine collected after a prostate massage before and after prostate biopsy, and from fecal samples collected before the biopsy. We sequenced DNA using the bacterial 16S rRNA high-throughput next-generation sequencing and analyzed changes in microbial profiles for taxonomy comparison between samples. RESULTS: Pre-biopsy urinary microbial profiles contained Lactobacillus and Staphylococcus bacteria. Post-biopsy urinary microbial profiles included lower levels of Lactobacillus and higher levels of Prevotella bacteria. Bacteroides bacteria were predominant in fecal samples. We identified two clustering patterns containing both pre- and post-biopsy urine samples. Cluster 1 had a urine cluster pattern that was distinct from fecal, whereas cluster 2 was similar to fecal. We observed two different modes of microbial changes, 11 patients had both of their urine (pre and post) samples associated with a particular cluster group, whereas others (n = 15) had movement between clusters 1 and 2 following the biopsy procedure. Four patient's post-biopsy urine microbial profiles clustered very tightly to the fecal microbial profile. CONCLUSIONS: We describe two models of change in the urinary tract microbiota after prostate biopsy using 16S RNA gene analysis. Further research to determine what controls changes in the urinary microbiota after prostate biopsy can help us understand why some patients are more susceptible to develop post-biopsy infections.

A prospective study to examine the association of the urinary and fecal microbiota with prostate cancer diagnosis after transrectal biopsy of the prostate using 16sRNA gene analysis.

INTRODUCTION: There is accumulating evidence that variations in the human microbiota may promote disease states including cancer. Our goal was to examine the association between urinary and fecal microbial profiles and the diagnosis of prostate cancer (PC) in patients undergoing transrectal biopsy of the prostate. MATERIALS AND METHODS: We extracted total DNA from urine and fecal samples collected before a prostate biopsy performed for elevated prostatic specific antigen in patients suspected of having PC. We then amplified the extracted DNA and sequenced it using bacterial 16S rRNA gene high-throughput next-generation sequencing platform, and analyzed microbial profiles for taxonomy comparing those patients diagnosed with PC with those who did not receive that diagnosis. RESULTS: We included 30 patients in our analysis (60 samples, one urine and one fecal per patient). The majority of patients with PC (10/14) had similar bacterial communities within their urinary sample profile and clustered separately than patients without cancer (n = 16). Differential analysis of the operational taxonomical units (OTUs) in urine samples revealed decreased abundance of several bacterial species in patients with prostate cancer. Analysis of the bacterial taxonomies of the fecal samples did not reveal any clustering in concordance with benign or malignant prostate biopsies. Patients who had a Gleason score (GS) of 6 (n = 11) were present in both urine bacterial community clusters, but patients with GS 7 or higher (n = 3) did not cluster tightly with non-cancer subjects. CONCLUSIONS: The urinary microbiota of patients with PC tends to cluster separately from those without this disease. Further research is needed to investigate the urinary microbiome potential of serving as a biomarker that could be used to improve the accuracy of pre-biopsy models predicting the presence of PC in post-biopsy tissue examination.

Effects of coconut oil on glycemia, inflammation, and urogenital microbial parameters in female Ossabaw mini-pigs.

Forty percent of American women are obese and at risk for type II diabetes, impaired immune function, and altered microbiome diversity, thus impacting overall health. We investigated whether obesity induced by an excess calorie, high fat diet containing hydrogenated fats, fructose, and coconut oil (HFD) altered glucose homeostasis, peripheral immunity, and urogenital microbial dynamics. We hypothesized that HFD would cause hyperglycemia, increase peripheral inflammation, and alter urogenital microbiota to favor bacterial taxonomy associated with inflammation. We utilized female Ossabaw mini-pigs to model a 'thrifty' metabolic phenotype associated with increased white adipose tissue mass. Pigs were fed HFD (~4570 kcal/pig/day) or lean (~2000 kcal/pig/day) diet for a total of 9 estrous cycles (~6 months). To determine the effect of cycle stage on cytokines and the microbiome, animals had samples collected during cycles 7 and 9 on certain days of the cycle: D1, 4, 8, 12, 16, 18. Vaginal swabs or cervical flushes assessed urogenital microbiota. Systemic fatty acids, insulin, glucose, and cytokines were analyzed. Pig weights and morphometric measurements were taken weekly. Obese pigs had increased body weight, length, heart and belly girth but similar glucose concentrations. Obese pigs had decreased cytokine levels (IL-1ß, TNF-α, IL-4, IL-10), arachidonic acid and plasma insulin, but increased levels of vaccenic acid. Obese pigs had greater urogenital bacterial diversity, including several taxa known for anti-inflammatory properties. Overall, induction of obesity did not induce inflammation but shifted the microbial communities within the urogenital tract to an anti-inflammatory phenotype. We postulate that the coconut oil in the HFD oil may have supported normal glucose homeostasis and modulated the immune response, possibly through regulation of microbial community dynamics and fatty acid metabolism. This animal model holds promise for the study of how different types of obesity and high fat diets may affect metabolism, immune phenotype, and microbial dynamics.

The Intestinal Microbiota Influences Campylobacter jejuni Colonization and Extraintestinal Dissemination in Mice.

Campylobacter jejuni is a leading cause of human foodborne gastroenteritis worldwide. The interactions between this pathogen and the intestinal microbiome within a host are of interest as endogenous intestinal microbiota mediates a form of resistance to the pathogen. This resistance, termed colonization resistance, is the ability of commensal microbiota to prevent colonization by exogenous pathogens or opportunistic commensals. Although mice normally demonstrate colonization resistance to C. jejuni, we found that mice treated with ampicillin are colonized by C. jejuni, with recovery of Campylobacter from the colon, mesenteric lymph nodes, and spleen. Furthermore, there was a significant reduction in recovery of C. jejuni from ampicillin-treated mice inoculated with a C. jejuni virulence mutant (ΔflgL strain) compared to recovery of mice inoculated with the C. jejuni wild-type strain or the C. jejuni complemented isolate (ΔflgL/flgL). Comparative analysis of the microbiota from nontreated and ampicillin-treated CBA/J mice led to the identification of a lactic acid-fermenting isolate of Enterococcus faecalis that prevented C. jejuni growth in vitro and limited C. jejuni colonization of mice. Next-generation sequencing of DNA from fecal pellets that were collected from ampicillin-treated CBA/J mice revealed a significant decrease in diversity of operational taxonomic units (OTUs) compared to that in control (nontreated) mice. Taken together, we have demonstrated that treatment of mice with ampicillin alters the intestinal microbiota and permits C. jejuni colonization. These findings provide valuable insights for researchers using mice to investigate C. jejuni colonization factors, virulence determinants, or the mechanistic basis of probiotics.