A pilot study using unique targeted testing of the urogenital microbiome has potential as a predictive test during IVF for implantation outcome.

PURPOSE: This pilot study aimed to develop a methodology characterising the urogenital microbiome as a predictive test in the IVF workup. METHODS: Using unique custom qPCRs, we tested for the presence of specific microbial species from vaginal samples and First Catch Urines from the male. The test panel included a range of potential urogenital pathogens, STIs, 'favourable bacteria' (Lactobacillus spp.) and 'unfavourable bacteria' (anaerobes) reported to influence implantation rates. We tested couples attending Fertility Associates, Christchurch, New Zealand for their first round of IVF. RESULTS: We found that some microbial species affected implantation. The qPCR result was interpreted qualitatively using the Z proportionality test. Samples from women at the time of Embryo Transfer who did not achieve implantation had significantly higher percent of samples that were positive for Prevotella bivia and Staphylococcus aureus compared to women who did achieve implantation. DISCUSSION: The results provide evidence that most other microbial species chosen for testing had little functional effect on implantation rates. The addition of further microbial targets (yet to be determined) could be combined in this predictive test for vaginal preparedness on the day of embryo transfer. This methodology has a substantial advantage of being affordable and easily performed in any routine molecular laboratory. This methodology is most suitable as a foundation on which to develop a timely test of microbiome profiling. Using the indicators detected to have a significant influence, these results can be extrapolated. CONCLUSION: Using a rapid antigen test, a woman can self-sample prior to embryo transfer and obtain an indication of microbial species present which could influence implantation outcome.

Differential Expression of Steroid Hormone Receptors and Ten Eleven Translocation Proteins in Endometrial Cancer Cells.

Steroid hormones govern the complex, cyclic changes of the endometrium, predominantly through their receptors. An interplay between steroid hormones and epigenetic mechanisms controls the dynamic endometrial gene regulation. Abnormalities in expression of genes and enzymes associated with steroid hormone signaling, contribute to a disturbed hormonal equilibrium. Limited evidence suggests the involvement of TET (Ten Eleven Translocation)-mediated DNA hydroxymethylation in endometrial cancer, with some data on the use of TET1 as a potential prognostic and diagnostic biomarker, however the mechanisms guiding it and its regulation remains unexplored. This study aims to explore the changes in the expressions of TETs and steroid hormone receptors in response to estrogen and progesterone in endometrial cancer cells. Gene expression was examined using real-time PCR and protein expression was quantified using fluorescent western blotting in endometrial cancer cell lines (AN3 and RL95-2). Results indicate that TET1 and TET3 gene and protein expression was cell-specific in cancer cell-lines. Protein expression of TET1 was downregulated in AN3 cells, while TET1 and TET3 expressions were both upregulated in RL95-2 cells in response to estrogen-progesterone. Further, a decreased AR expression in AN3 cells and an increased ERα and ERß protein expressions in RL95-2 cells was seen in response to estrogen-progesterone. PR gene and protein expression was absent from both cancer cell-lines. Overall, results imply that expressions of steroid hormones, steroid-hormone receptors and TETs are co-regulated in endometrial cancer-cells. Further studies are needed to interpret how these mechanisms fit in with DNMTs and DNA methylation in regulating endometrial biology. Understanding the role of TETs and hydroxymethylation in steroid hormone receptor regulation is crucial to comprehend how these mechanisms work together in a broader context of epigenetics in the endometrium and its pathologies.

The vaginal microbiota and innate immunity after local excisional treatment for cervical intraepithelial neoplasia.

BACKGROUND: Vaginal microbiota (VMB) composition is altered in women with cervical intra-epithelial neoplasia (CIN) compared to healthy controls and is associated with disease progression. However, the impact of CIN excision on the VMB and innate immunity is not known. This observational study aims to explore the impact of CIN excision on the VMB, antimicrobial peptides (AMP) and proinflammatory cytokines. METHODS: We sampled 103 non-pregnant, premenopausal women at the time of excisional treatment for CIN and at their 6-month follow-up visit. A further 39 untreated controls with normal cytology were also sampled. We used metataxonomics to group vaginal swab samples into community state types (CSTs) and ELISA to quantify cytokine and AMP levels in matched vaginal secretions. Analyses were performed to compare the bacterial composition and immune analyte levels before and after CIN excision and in healthy controls. RESULTS: Women with CIN had significantly higher rates of Lactobacillus species depletion pre-treatment compared to healthy controls (CST IV 21/103, 20% vs 1/39, 3%, p = 0.0081). Excision did not change the VMB composition, with CST IV remaining significantly more prevalent after excision compared to untreated, healthy controls (CST IV 19/103, 20% vs 1/39, 3%, p = 0.0142). Prevotella bivia and Sneathia amnii were significantly higher in samples before treatment compared to untreated controls, and Prevotella bivia remained significantly higher amongst the treated, with less Lactobacillus crispatus compared to untreated controls. IL-1ß and IL-8 remained significantly elevated pre- (p < 0.0001 and p = 0.0014, respectively) and post-treatment (p < 0.0001 and p = 0.0035, respectively) compared to untreated controls. Levels of human beta-defensin-1 and secretory leukocyte protease inhibitor were both significantly reduced following CIN excision (p < 0.0001); however, their levels remained lower than controls post-treatment. CONCLUSIONS: Women with CIN have an increased prevalence of Lactobacillus sp. depletion, high-diversity VMB composition, and higher levels of proinflammatory cytokines and AMPs compared to normal controls. Surgical excision of the disease reduces levels of vaginal AMPs but does not alter VMB composition or cytokine levels. These findings suggest that women with CIN have an inherent predisposition to a high-diversity proinflammatory environment that is not corrected by disease excision. The failure to re-establish a Lactobacillus-enriched CST may explain why women remain at high risk of pre-invasive and invasive disease recurrence.

Histone acetylation and the role of histone deacetylases in normal cyclic endometrium.

Histone acetylation is a critical epigenetic modification that changes chromatin architecture and regulates gene expression by opening or closing the chromatin structure. It plays an essential role in cell cycle progression and differentiation. The human endometrium goes through cycles of regeneration, proliferation, differentiation, and degradation each month; each phase requiring strict epigenetic regulation for the proper functioning of the endometrium. Aberrant histone acetylation and alterations in levels of two acetylation modulators - histone acetylases (HATs) and histone deacetylases (HDACs) - have been associated with endometrial pathologies such as endometrial cancer, implantation failures, and endometriosis. Thus, histone acetylation is likely to have an essential role in the regulation of endometrial remodelling throughout the menstrual cycle.

Expression and steroid hormone regulation of TETs and DNMTs in human endometrium.

DNA methyltransferases (DNMTs) and ten-eleven translocation proteins (TETs) facilitate methylation and hydroxymethylation of DNA, respectively. DNMTs are widely studied with conflicting results on their regulation in the endometrium. While the role of TETs in the endometrium remains relatively unexplored. Deregulated expression of TETs and DNMTs are associated with endometrial pathologies. The aim of this study is to characterize the temporal TET expression in endometrium and to determine the hormonal regulation of TETs in comparison to DNMTs. mRNA expressions were quantified by real-time PCR in endometrial tissues from cycling women and localization was determined by immunohistochemistry. Hormonal regulation was investigated in endometrial epithelial and stromal cell lines following a 24 and 48 h treatment cycle. TET1 and 3 mRNA expressions were significantly upregulated in the mid-secretory phase. TET protein expression was ubiquitous in endometrial epithelium throughout the menstrual cycle except during the late-secretory phase, while stromal staining was scattered. TET1 mRNA was significantly upregulated in response to estrogen in stromal cells. Transcriptions of all three TETs were induced in response to progesterone treatment in epithelial cells. Only DNMT3b in epithelial cells and DNMT1 in stromal cells were significantly upregulated upon 24-h estrogen exposure following a significant decrease of DNMT1 when treated with 24 h of estrogen and progesterone. This study suggests that TETs are expressed in a cell-specific, dynamic manner in the endometrium and are responsive to steroid hormones. Investigating the role of TETs individually and with respect to DNMTs, will help to elucidate gene regulatory mechanisms in endometrial biology and pathologies.

Could DNA hydroxymethylation be crucial in influencing steroid hormone signaling in endometrial biology and endometriosis?

Endometriosis affects 10% of reproductive-aged women. It is characterized by the growth of the endometrium, outside the uterus and is associated with infertility and chronic abdominal pain. Lack of noninvasive diagnostic tools and early screening tests results in delayed treatment and subsequently increased disease severity. Endometriosis is a disease associated with a deregulated hormonal response, therefore, understanding the molecular mechanisms that govern this hormonal interplay is of paramount importance. DNA methylation is an epigenetic mark that regulates gene expression and is often associated with genes that code for steroid receptors and enzymes associated with estrogen synthesis and metabolism in endometriosis. DNA hydroxymethylation, which is structurally similar to methylation but functionally different, is a biologically critical mechanism that is also known to regulate gene expression. Ten Eleven Translocation (TET) proteins mediate hydroxymethylation. However, the role of DNA hydroxymethylation or TETs in the endometrium remains relatively unexplored. Currently, the "gold standard" technique used to study methylation patterns is bisulfite genomic sequencing. This technique also detects hydroxymethylation but fails to distinguish between the two, thereby limiting our understanding of these two processes. The presence of TETs in the male and female reproductive tract and its contribution to endometrial cancer makes it an important factor to study in endometriosis. This review summarizes the role of DNA methylation in aberrant steroid hormone signaling and hypothesizes that hydroxymethylation could be a factor influencing hormonal instability seen in endometriosis.

Comparison of vaginal microbiota sampling techniques: cytobrush versus swab.

Evidence suggests the vaginal microbiota (VM) may influence risk of persistent Human Papillomavirus (HPV) infection and cervical carcinogenesis. Established cytology biobanks, typically collected with a cytobrush, constitute a unique resource to study such associations longitudinally. It is plausible that compared to rayon swabs; the most commonly used sampling devices, cytobrushes may disrupt biofilms leading to variation in VM composition. Cervico-vaginal samples were collected with cytobrush and rayon swabs from 30 women with high-grade cervical precancer. Quantitative PCR was used to compare bacterial load and Illumina MiSeq sequencing of the V1-V3 regions of the 16S rRNA gene used to compare VM composition. Cytobrushes collected a higher total bacterial load. Relative abundance of bacterial species was highly comparable between sampling devices (R2 = 0.993). However, in women with a Lactobacillus-depleted, high-diversity VM, significantly less correlation in relative species abundance was observed between devices when compared to those with a Lactobacillus species-dominant VM (p = 0.0049). Cytobrush and swab sampling provide a comparable VM composition. In a small proportion of cases the cytobrush was able to detect underlying high-diversity community structure, not realized with swab sampling. This study highlights the need to consider sampling devices as potential confounders when comparing multiple studies and datasets.