Potential contribution of the uterine microbiome in the development of endometrial cancer.

BACKGROUND: Endometrial cancer studies have led to a number of well-defined but mechanistically unconnected genetic and environmental risk factors. One of the emerging modulators between environmental triggers and genetic expression is the microbiome. We set out to inquire about the composition of the uterine microbiome and its putative role in endometrial cancer. METHODS: We undertook a study of the microbiome in samples taken from different locations along the female reproductive tract in patients with endometrial cancer (n = 17), patients with endometrial hyperplasia (endometrial cancer precursor, n = 4), and patients afflicted with benign uterine conditions (n = 10). Vaginal, cervical, Fallopian, ovarian, peritoneal, and urine samples were collected aseptically both in the operating room and the pathology laboratory. 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. Microbiota data were summarized using both α-diversity to reflect species richness and evenness within bacterial populations and ß-diversity to reflect the shared diversity between bacterial populations. Statistical significance was determined through the use of multiple testing, including the generalized mixed-effects model. RESULTS: The microbiome sequencing (16S rDNA V3-V5 region) revealed that the microbiomes of all organs (vagina, cervix, Fallopian tubes, and ovaries) are significantly correlated (p < 0.001) and that there is a structural microbiome shift in the cancer and hyperplasia cases, distinguishable from the benign cases (p = 0.01). Several taxa were found to be significantly enriched in samples belonging to the endometrial cancer cohort: Firmicutes (Anaerostipes, ph2, Dialister, Peptoniphilus, 1-68, Ruminococcus, and Anaerotruncus), Spirochaetes (Treponema), Actinobacteria (Atopobium), Bacteroidetes (Bacteroides and Porphyromonas), and Proteobacteria (Arthrospira). Of particular relevance, the simultaneous presence of Atopobium vaginae and an uncultured representative of the Porphyromonas sp. (99 % match to P. somerae) were found to be associated with disease status, especially if combined with a high vaginal pH (>4.5). CONCLUSIONS: Our results suggest that the detection of A. vaginae and the identified Porphyromonas sp. in the gynecologic tract combined with a high vaginal pH is statistically associated with the presence of endometrial cancer. Given the documented association of the identified microorganisms with other pathologies, these findings raise the possibility of a microbiome role in the manifestation, etiology, or progression of endometrial cancer that should be further investigated.

Emphysematous pyometra secondary to Enterococcus avium infection in a dog.

A 5-year-old female intact Mastiff dog was presented with a history of vaginal discharge for 1 day. Physical examination revealed a sanguineo-purulent vaginal discharge and systemic inflammatory response syndrome. Abdominal radiographs showed several dilated and gas- filled tubular loops. The differential diagnoses included emphysematous pyometra or small intestinal mechanical ileus. Surgical exploration of the abdomen demonstrated a severely dilated and gas-filled uterus, and emphysematous pyometra was confirmed. The patient's clinical signs resolved after ovariohysterectomy. Histopathology revealed mild endometrial cystic hyperplasia with infiltration of inflammatory cells in the superficial endometrial epithelia. Enterococcus avium, an α-hemolytic gram-positive coccus, was isolated from the uterus. This paper highlights the radiographic features of emphysematous pyometra and a pathogen that has never been reported to be associated with canine pyometra previously.

Kynurenic acid in plasma and endometrium in bitches with pyometra.

Kynurenic acid (KYNA) is produced enzymatically in humans and animals from kynurenine. Reports concerning changes of kynurenine metabolism during inflammation are available in the literature. Pyometra is a pathological condition characterized by the accumulation of pus in the uterine lumen and bacterial infection. The objective of the study was to compare the serum and endometrial KYNA concentrations in healthy bitches and those with pyometra. KYNA was determined by means of high-performance liquid chromatography with fluorometric detection. The serum content of KYNA in bitches with pyometra was significantly higher than in healthy bitches. The KYNA content in the endometrium of bitches with pyometra was higher, yet the difference was not statistically significant. Our result indicates that determination of KYNA might be a marker of pyometra in bitches.

Gene transcription of TLR2, TLR4, LPS ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia-pyometra complex.

Escherichia coli (E. coli) is the most frequent bacterium isolated in cases of cystic endometrial hyperplasia-pyometra complex, the most frequent endometrial disorder in the bitch. Toll-like receptors (TLRs) play an essential role in the innate immune system. The aim of this study was to compare transcription of genes encoding TLR2, TLR4 and LPS ligands (CD14, MD-2, LBP), prostaglandin synthesis enzymes (COX1, COX2, PGES1 and PGFS), and to compare COX1 and COX2 protein expression and PGE(2) and PGF(2alpha) endometrial content in the endometrium of canine diestrous uteri with (n=7) or without (n=7) pyometra. All cases of pyometra were hyperplastic and E. coli was the only isolated bacteria, while diestrous normal uteri did not present signs of cystic endometrial hyperplasia and were negative for bacteriology. Except for COX1, transcription of all genes was significantly higher in pyometra than in normal endometria. COX1 protein was observed in both normal and pyometra uteri, but COX2 protein was only present in pyometra cases. Endometrial PGE(2) and PGF(2alpha) content were significantly higher in pyometra than in normal diestrous endometria. In conclusion, data obtained in this study provides evidence that pyometra-isolated E. coli induces the up-regulation of TLR2 and TLR4 genes in the canine diestrous endometrium. This up-regulation, which is probably the result of the stimulation by LPS and lipoprotein E. coli constituents, leads to the endometrial up-regulation of PG synthesis genes. This, in turn, results in a higher endometrial concentration of PGE(2) and PGF(2alpha), which may further regulate the local inflammatory response.

A model for cystic endometrial hyperplasia/pyometra complex in the bitch.

The objective of this study was to develop a reliable model for the study of the cystic endometrial hyperplasia and pyometra complex (CEH/P) in the bitch. Greyhound bitches (n = 15) were ovariectomised and allocated into three groups (Group 1, n = 5; Group 2, n = 5; Group 3, n = 10, including 5 used from Group 1). Simulated proestrus, estrus and diestrus were induced by treatment with estradiol benzoate and megestrol acetate. The duration of cervical opening during estrus was determined by the intra-vaginal infusion of radio-opaque medium and subsequent radiography of the uterus (Group 1). One milliliter of a culture of Escherichia coli (with five uro-pathogenic virulence factors as identified by PCR: pap, sfa, hlyA, cnf1 and fim) was inoculated intra-vaginally daily throughout the simulated estrus (Group 2). One milliliter of the culture (n = 6) or sterile Luria-Bertani broth (n = 4) was introduced directly into the uterus on simulated diestrus Days 8 or 12 (Group 3). Necropsies were performed 12 and 7-14 days after the inoculation (Groups 2 and 3). The cervix remained open throughout the duration of simulated estrus (5-6 days) in four out of five bitches, and for a shorter duration (3 days of a 6-day estrus period) in one bitch (Group 1). CEH/P was induced by inoculation of bacteria into the uterus (10/10 bitches) but not into the vagina (0/5 bitches), (P = 0.003). A model for the study of CEH/P has been validated.

The effects of endometrial scarification on uterine steroid receptors, bacterial flora and histological structure in the bitch.

Following laparotomy, the endometrium of six nulliparous Beagle bitches was scarified at the base of one uterine horn during early metoestrus, when the peripheral plasma P(4) concentration was >10 ng/ml; intrauterine swabs were taken at the same time for bacteriological culture. Twenty-one days later, a bilateral ovariohysterectomy was performed and segments of the scarified and non-scarified parts of the tubular genital tract removed; at the same time, swabs were taken from the uterine lumen. Tissue samples were collected and examined for histopathological structure, and the presence of nuclear oestrogen (ER) and progesterone (PR) receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score). All uterine swabs were sterile, and in three of the six bitches there were noticeable changes with distension of the uterine lumen with secretions and debris and distension of the endometrial gland ducts of the scarified uterine segment. There were no statistically significant differences in the H-scores of ER or PR between scarified and non-scarified segments, except for PR H-scores in the glandular epithelium where the values for the scarified were significantly higher than for the non-scarified endometrium (mean+/-S.E.M. is 129.9+/-22.8 versus 59.5+/-12.6; P<0.05). Thus, trauma can modify the structure of the endometrium and the characteristics of the PR. Whether changes in PR expression are involved in the pathogenesis of CEH/pyometra in the bitch could not be ascertained from this study.

Uterine bacterial flora and uterine lesions in bitches with cystic endometrial hyperplasia (pyometra).

Thirty-eight complete genital tracts were obtained fresh from local veterinary practices after bitches had been ovariohysterectomised for the treatment of cystic endometrial hyperplasia (pyometra). The aerobic and anaerobic culture of swabs obtained aseptically from the uterine lumen demonstrated the presence of Escherichia coli in 28 of the 29 cases from which bacteria were isolated and, from 25, a pure culture was obtained. Sixteen different serotypes were identified from 24 genital tracts; 032K+ CNF+ was isolated from four of 16 cases and 04K-; from three of 16 cases. Cytotoxin necrotising factors (CNFs) were identified in seven of the 16 serotypes; they included 02, 04, 06, 022, 025, 045 and 075, which except for 06 and 045 were haemolytic. Strains 04K- and 07K+ completely destroyed the integrity of the endometrial epithelium whereas 088K+ and 088K- had the least effect. The scores for the reduction in the integrity of the endometrial epithelium and the degree of infiltration of inflammatory cells were greater in the presence of CNF 1.

Effects of energy restriction on mouse mammary tumor virus mRNA levels in mammary glands and uterus and on uterine endometrial hyperplasia and pituitary histology in C3H/SHN F1 mice.

We investigated the effects of energy restriction on the pituitary-ovarian axis and on a hormone responsive gene, the mouse mammary tumor virus (MMTV). Female C3H/SHN F1-hybrid mice, known to display a high incidence of mammary tumors, ate an energy-restricted diet (48 kcal/wk) or a control diet (95 kcal/wk) beginning at the time of weaning. By 67 wk of age, 12 of 32 mice in the control group, but none of the 33 mice in the energy-restricted group, had developed mammary tumors. Six tumor-free mice from each group were studied in detail at 67 wk of age. All six tumor-free control mice, but none of the six energy-restricted mice, showed uterine endometrial hyperplasia at autopsy. Mice subjected to energy restriction did not display an estrous cycle. The average levels of MMTV mRNA in mammary glands and uteri were strongly reduced by energy restriction. MMTV mRNA levels in mammary glands from control mice were two orders of magnitude lower than those in mammary tumors. Energy restriction lowered the percentage of pituitary mammatropes and suppressed proliferation of mammatropes with advancing age. Energy restriction thus appeared to inhibit endometrial hyperplasia and to decrease MMTV production at the mRNA level in the mammary glands and in the uterus. These effects may be a consequence of hormonal changes originating at the pituitary-ovarian axis.