Josephine Drury

Stage‐specific embryonic antigen‐1 (SSEA‐1) ⁺ endometrial epithelial cells (EECs) assume the postulated stem/progenitor cell niche within the human endometrium. Previous studies have demonstrated that isolated SSEA‐1 ⁺ cells have a higher capacity to generate organoids in a three‐dimensional matrix, a lower steroid hormone receptor expression, and higher telomerase activity with longer telomere lengths. Here, we present the transcriptomic profile of isolated SSEA‐1 ⁺ EECs from eight endometrial biopsies compared to SSEA‐1 ⁻ EECs. Transcriptome and pathway analysis indicate that SSEA‐1 ⁺ EECs play an important role in endometrial regeneration, remodeling and neovascularization as expected from a basal progenitor population. We show that SSEA‐1 ⁺ EECs play a role in endometrial tissue homeostasis and tumor suppression, and bioinformatically identify potential upstream regulators such as SPDEF and TGFB1, which may be involved in these mechanisms. In vitro EEC organoid models also demonstrate SSEA‐1 ⁺ EECs to exhibit estrogen responsive proliferation evidenced by stronger immunostaining for progesterone receptor and Ki‐67. Our data further suggest a more quiescent, less hormone responsive phenotype for SSEA‐1 ⁺ EECs that co‐localize to SOX9 ⁺ EECs within in silico analysis, thus validating previous studies.

Many women report embarrassment as the cause for their avoidance of routine gynaecological screening appointments. Methods of self-collection of bio samples would perhaps encourage women to participate in routine screening programs. The vaginal microbiome plays a key role in women’s health and reproductive function. Microbial disturbances can result in the loss of lactobacillus dominance, also known as dysbiosis, associated with an increased risk of contracting sexually transmitted infections (STIs), pregnancy complications and infertility. Our primary aim was to determine if vaginal microbiome screening results are comparable between two methods for self-collected sample acquisition: tampons and lower vaginal swabs (LVSs). Secondary aims included the assessment of the effect of pre-analytic storage on the data (to streamline processing), the prevalence of dysbiosis and the acceptability of the tampons to the participants. Statistical analysis revealed no significant difference in the microbiome data, from tampons versus LVSs or fresh versus frozen samples. The prevalence of dysbiosis in this population of healthy volunteers was 42.9%. The questionnaire data revealed that 52.4% of volunteers use tampons every period, and the majority of volunteers rated the tampons as 5 on a 1–5 Likert scale regarding their perceived comfort using tampons. All (100%) of volunteers were happy to provide a tampon as a sample for testing. The findings from this study show that tampons and LVSs were comparable when analysing the vaginal microbiome, with potential superiority of the tampon with regard to patient acceptability. Self-collection of vaginal secretions for gynaecological screening using tampons warrants further research as this could change the screening landscape, ensuring wider participation and increasing efficacy.

Endometrial cancer (EC) is the most common gynaecological malignancy. Nucleolin (NCL) is involved in rDNA transcription, cell proliferation, and apoptosis, with high expression associated with worse overall survival (OS) in other adenocarcinomas. Our aims were to assess NCL gene and protein expression and explore the differential expression of NCL-associated genes (NAGs) in endometrial carcinogenesis. Endometrial samples were obtained from 157 women to include healthy, hyperplastic (EH), EC, and metastatic groups. RT-qPCR and immunohistochemistry were employed to assess NCL gene and protein levels. In silico analysis of NAGs in TCGA and GEO datasets was performed, with the prognostic value determined via Human Protein Atlas. NCL mRNA level of EC was lower than in healthy post-menopausal endometrium (p < 0.01). EH samples had lower NCL immuno-expression scores than healthy pre-menopausal (p < 0.001), benign post-menopausal (p < 0.01), and EC (p < 0.0001) samples. Metastatic lesions demonstrated higher NCL quick scores than primary tissue (p = 0.04). Higher NCL Immuno quick scores carried a worse OS in high-grade EC (p = 0.01). Interrogating Uterine Corpus Endometrial Carcinoma (TCGA-UCEC) and Uterine Carcinosarcoma (TCGA-UCS) cohorts revealed NCL to be the most highly upregulated gene in carcinosarcoma, with S100A11, LMNB2, RERG, E2F1 and CCNA2 representing key dysregulated NAGs in EC. Since NCL is implicated in transforming hyperplastic glands into cancer, with further involvement in metastasis, it is suggested to be a promising target for better-informed diagnosis, risk stratification, and management of EC.

Risk of relapse of endometrial cancer (EC) after surgical treatment is 13% and recurrent disease carries a poor prognosis. Research into prognostic indicators is essential to improve EC management and outcome. “Immortality” of most cancer cells is dependent on telomerase, but the role of associated proteins in the endometrium is poorly understood. The Cancer Genome Atlas data highlighted telomere/telomerase associated genes (TTAGs) with prognostic relevance in the endometrium, and a recent in silico study identified a group of TTAGs and proteins as key regulators within a network of dysregulated genes in EC. We characterise relevant telomere/telomerase associated proteins (TTAPs) NOP10, NHP2, NOP56, TERF1, TERF2 and TERF2IP in the endometrium using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC). qPCR data demonstrated altered expression of multiple TTAPs; specifically, increased NOP10 (p = 0.03) and reduced NHP2 (p = 0.01), TERF2 (p = 0.01) and TERF2IP (p < 0.003) in EC relative to post-menopausal endometrium. Notably, we report reduced NHP2 in EC compared to post-menopausal endometrium in qPCR and IHC (p = 0.0001) data; with survival analysis indicating high immunoscore is favourable in EC (p = 0.0006). Our findings indicate a potential prognostic role for TTAPs in EC, particularly NHP2. Further evaluation of the prognostic and functional role of the examined TTAPs is warranted to develop novel treatment strategies.

Recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) are distressing conditions without effective treatments. The luminal epithelium (LE) is integral in determining receptivity of the endometrium, whereas functionalis glands and stroma aid in nurturing early embryo development. Calcium signalling pathways are known to be of vital importance to embryo implantation and pregnancy establishment, and anterior gradient protein 3 (AGR3) and S100 calcium-binding protein P (S100P) are involved with these pathways. We initially examined 20 full-thickness endometrial biopsies from premenopausal women across the menstrual cycle to characterize levels of AGR3 protein in each endometrial sub-region at the cellular level. A further 53 endometrial pipelle biopsies collected in the window of implantation were subsequently assessed to determine differential endometrial AGR3 and S100P levels relevant to RIF (n = 13) and RPL (n = 10) in comparison with parous women (n = 30) using immunohistochemistry. Significantly higher AGR3 and S100P immunostaining was observed in ciliated cells of the LE of women with recurrent reproductive failure compared with parous women, suggesting aberrant subcellular location-associated pathophysiology for these conditions. The nuclear localisation of S100P may allow transcriptional regulatory function, which is necessary for implantation of a viable pregnancy. Further work is thus warranted to assess their utility as diagnostic/therapeutic targets.

Telomeres are transcribed as long non-coding RNAs called TERRAs (Telomeric repeat containing RNA) that participate in a variety of cellular regulatory functions. High telomerase activity (TA) is associated with endometrial cancer (EC). This study aimed to examine the levels of three TERRAs, transcribed at chromosomes 1q-2q-4q-10q-13q-22q, 16p and 20q in healthy (n = 23) and pathological (n = 24) human endometrium and to examine their association with cellular proliferation, TA and telomere lengths. EC samples demonstrated significantly reduced levels of TERRAs for Chromosome 16p (Ch-16p) (p < 0.002) and Chromosome 20q (Ch-20q) (p = 0.0006), when compared with the postmenopausal samples. No significant correlation was found between TERRA levels and TA but both Ch-16p and Ch-20q TERRA levels negatively correlated with the proliferative marker Ki67 (r = −0.35, p = 0.03 and r = −0.42, p = 0.01 respectively). Evaluation of single telomere length analysis (STELA) at XpYp telomeres demonstrated a significant shortening in EC samples when compared with healthy tissues (p = 0.002). We detected TERRAs in healthy human endometrium and observed altered individual TERRA-specific levels in malignant endometrium. The negative correlation of TERRAs with cellular proliferation along with their significant reduction in EC may suggest a role for TERRAs in carcinogenesis and thus future research should explore TERRAs as potential therapeutic targets in EC.

Endometrial cancer (EC) is the commonest gynaecological malignancy. Current prognostic markers are inadequate to accurately predict patient survival, necessitating novel prognostic markers, to improve treatment strategies. Telomerase has a unique role within the endometrium, whilst aberrant telomerase activity is a hallmark of many cancers. The aim of the current in silico study is to investigate the role of telomere and telomerase associated genes and proteins (TTAGPs) in EC to identify potential prognostic markers and therapeutic targets. Analysis of RNA-seq data from The Cancer Genome Atlas identified differentially expressed genes (DEGs) in EC (568 TTAGPs out of 3467) and ascertained DEGs associated with histological subtypes, higher grade endometrioid tumours and late stage EC. Functional analysis demonstrated that DEGs were predominantly involved in cell cycle regulation, while the survival analysis identified 69 DEGs associated with prognosis. The protein-protein interaction network constructed facilitated the identification of hub genes, enriched transcription factor binding sites and drugs that may target the network. Thus, our in silico methods distinguished many critical genes associated with telomere maintenance that were previously unknown to contribute to EC carcinogenesis and prognosis, including NOP56, WFS1, ANAPC4 and TUBB4A. Probing the prognostic and therapeutic utility of these novel TTAGP markers will form an exciting basis for future research.

Several diseases of prematurity are thought to be related to oxidative injury and many of the available markers are unsatisfactory. An assay was developed using HPLC with electrochemical detection for the quantitation of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) as a proposed indicator for oxygen-derived free radical injury to DNA in preterm infants. A median value of 3.79 pmol/mol creatinine was obtained for normal children (2–15 years old, n = 14). Urinary 8-OHdG excretion in neonates ranged from 0–99μmol/mol creatinine. There were no gestation or birthweight related differences in urinary 8-OHdG, and no correlation with urinary malondialdehyde. Mean 8-OHdG excretion increased with postnatal age (r= 0.80, p < 0.0001, n = 15), mirroring the growth velocity curve. These changes could also be due to changes in the activity of the enzyme responsible for 8-OHdG excision. Urinary 8-OHdG levels are unlikely to accurately reflect oxygen derived free radical activity given the strength of the relationship with growth.