Physiology of the Endometrium and Regulation of Menstruation.

The physiological functions of the uterine endometrium (uterine lining) are preparation for implantation, maintenance of pregnancy if implantation occurs, and menstruation in the absence of pregnancy. The endometrium thus plays a pivotal role in reproduction and continuation of our species. Menstruation is a steroid-regulated event, and there are alternatives for a progesterone-primed endometrium, i.e., pregnancy or menstruation. Progesterone withdrawal is the trigger for menstruation. The menstruating endometrium is a physiological example of an injured or "wounded" surface that is required to rapidly repair each month. The physiological events of menstruation and endometrial repair provide an accessible in vivo human model of inflammation and tissue repair. Progress in our understanding of endometrial pathophysiology has been facilitated by modern cellular and molecular discovery tools, along with animal models of simulated menses. Abnormal uterine bleeding (AUB), including heavy menstrual bleeding (HMB), imposes a massive burden on society, affecting one in four women of reproductive age. Understanding structural and nonstructural causes underpinning AUB is essential to optimize and provide precision in patient management. This is facilitated by careful classification of causes of bleeding. We highlight the crucial need for understanding mechanisms underpinning menstruation and its aberrations. The endometrium is a prime target tissue for selective progesterone receptor modulators (SPRMs). This class of compounds has therapeutic potential for the clinical unmet need of HMB. SPRMs reduce menstrual bleeding by mechanisms still largely unknown. Human menstruation remains a taboo topic, and many questions concerning endometrial physiology that pertain to menstrual bleeding are yet to be answered.

Increased risk of HPV-associated genital cancers in men and women as a consequence of pre-invasive disease.

To assess the excess risk of HPV-associated cancer (HPVaC) in two at-risk groups-women with a previous diagnosis of high grade cervical intraepithelial neoplasia (CIN3) and both men and women treated for non-cervical pre-invasive anogenital disease. All CIN3 cases diagnosed in 1989-2015 in Scotland were extracted from the Scottish cancer registry (SMR06). All cases of pre-invasive penile, anal, vulval, and vaginal disease diagnosed in 1990-2015 were identified within the NHS pathology databases in the two largest NHS health boards in Scotland. Both were linked to SMR06 to extract subsequent incidence of HPVaC following the diagnosis of CIN3 or pre-invasive disease. Standardised incidence ratios were calculated for the risk of acquiring HPVaC for the two at-risk groups compared to the general Scottish population. Among 69,714 females in Scotland diagnosed with CIN3 (890,360.9 person-years), 179 developed non-cervical HPVaC. CIN3 cases were at 3.2-fold (95% CI: 2.7 to 3.7) increased risk of developing non-cervical HPVaC, compared to the general female population. Among 1,235 patients diagnosed with non-cervical pre-invasive disease (9,667.4 person-years), 47 developed HPVaC. Individuals with non-cervical pre-invasive disease had a substantially increased risk of developing HPVaC - 15.5-fold (95% CI: 11.1 to 21.1) increased risk for females and 28-fold (11.3 to 57.7) increased risk for males. We report a significant additional risk of HPV-associated cancer in those have been diagnosed with pre-invasive HPV-associated lesions including but not confined to the cervix. Uncovering the natural history of pre-invasive disease has potential for determining screening, prevention and treatment.

High EMSY expression defines a BRCA-like subgroup of high-grade serous ovarian carcinoma with prolonged survival and hypersensitivity to platinum.

BACKGROUND: Approximately half of high-grade serous ovarian carcinomas (HGSOCs) demonstrate homologous recombination repair (HR) pathway defects, resulting in a distinct clinical phenotype comprising hypersensitivity to platinum, superior clinical outcome, and greater sensitivity to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors. EMSY, which is known to be amplified in breast and ovarian cancers, encodes a protein reported to bind and inactivate BRCA2. Thus, EMSY overexpression may mimic BRCA2 mutation, resulting in HR deficiency. However, to our knowledge, the phenotypic consequences of EMSY overexpression in HGSOC patients has not been explored. METHODS: Here we investigate the impact of EMSY expression on clinical outcome and sensitivity to platinum-based chemotherapy using available data from transcriptomically characterized HGSOC cohorts. RESULTS: High EMSY expression was associated with better clinical outcome in a cohort of 265 patients with HGSOC from Edinburgh (overall survival multivariable hazard ratio, 0.58 [95% CI, 0.38-0.88; P = .011] and progression-free survival multivariable hazard ratio, 0.62 [95% CI, 0.40-0.96; P = .030]). Superior outcome also was demonstrated in the Medical Research Council ICON7 clinical trial and multiple publicly available data sets. Patients within the Edinburgh cohort who had high EMSY expression were found to demonstrate greater rates of complete response to multiple platinum-containing chemotherapy regimens (radiological complete response rate of 44.4% vs 12.5% at second exposure; P = .035) and corresponding prolonged time to disease progression (median, 151.5 days vs 60.5 days after third platinum exposure; P = .004). CONCLUSIONS: Patients with HGSOCs demonstrating high EMSY expression appear to experience prolonged survival and greater platinum sensitivity, reminiscent of BRCA-mutant cases. These data are consistent with the notion that EMSY overexpression may render HGSOCs HR deficient.

Expression of glycolytic enzymes in ovarian cancers and evaluation of the glycolytic pathway as a strategy for ovarian cancer treatment.

BACKGROUND: Novel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components (GLUT1, HKII, PKM2, LDHA) of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors. METHODS: Expression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents. RESULTS: Mean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway (STF31, IOM-1190, 3PO and oxamic acid) attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth. CONCLUSIONS: The findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.

Uterine NK cells regulate endometrial bleeding in women and are suppressed by the progesterone receptor modulator asoprisnil.

Uterine NK cells (uNK) play a role in the regulation of placentation, but their functions in nonpregnant endometrium are not understood. We have previously reported suppression of endometrial bleeding and alteration of spiral artery morphology in women exposed to asoprisnil, a progesterone receptor modulator. We now compare global endometrial gene expression in asoprisnil-treated versus control women, and we demonstrate a statistically significant reduction of genes in the IL-15 pathway, known to play a key role in uNK development and function. Suppression of IL-15 by asoprisnil was also observed at mRNA level (p < 0.05), and immunostaining for NK cell marker CD56 revealed a striking reduction of uNK in asoprisnil-treated endometrium (p < 0.001). IL-15 levels in normal endometrium are progesterone-responsive. Progesterone receptor (PR) positive stromal cells transcribe both IL-15 and IL-15RA. Thus, the response of stromal cells to progesterone will be to increase IL-15 trans-presentation to uNK, supporting their expansion and differentiation. In asoprisnil-treated endometrium, there is a marked downregulation of stromal PR expression and virtual absence of uNK. These novel findings indicate that the IL-15 pathway provides a missing link in the complex interplay among endometrial stromal cells, uNK, and spiral arteries affecting physiologic and pathologic endometrial bleeding.

Selective progesterone receptor modulators.

PURPOSE OF REVIEW: Review of recent data from clinical trials and descriptions of endometrial morphology with administration of selective progesterone receptor modulators (SPRMs). RECENT FINDINGS: Recent reports concerning administration of SPRMs, specifically the efficacy of ulipristal acetate in reducing fibroid size and rapid control of menstrual blood loss, have renewed clinical interest in this class of compound. Histological data from studies with SPRMs report that this class of drugs is associated with progesterone receptor modulator-associated endometrial changes. Data on mechanisms of action are lacking. The antagonistic progesterone effect of SPRMs has shown promising results in animal studies with endometriosis. Sex steroid receptor effects of PRMs outside the reproductive tract raise the potential for use in neurology and oncology, and although there are several randomized trials in these areas, there are limited small studies published to date. SUMMARY: The SPRM ulipristal acetate is an effective treatment for preoperative treatment of fibroids and a reliable emergency contraceptive. This class of compounds holds the potential for long-term effective medical management of fibroids and may have utility in the management of other sex steroid-dependent conditions.

A 39-week oral toxicity study of ulipristal acetate in cynomolgus monkeys.

Ulipristal acetate (UPA) is a novel Progesterone Receptor Modulator (PRM) and registered for the pre-operative treatment of symptomatic uterine fibroids during 3months. In a study which assessed the potential toxicity of UPA in female cynomolgus monkeys following daily oral administration of 1, 5, or 25mg/kg for 39weeks, UPA was well tolerated with dose-dependent macroscopic and microscopic observations limited to the uterus and oviducts. These findings were considered to be related to the pharmacological action of UPA and showed evidence of partial reversibility. Findings in the endometrium were similar to PRM-associated-endometrial-changes (PAEC) described in PRM-treated women. No adverse effects were found that would raise concerns about potential pre-malignancy. Although the translation of these findings to human is limited by the small study size and species differences, these results from animals chronically exposed to up to 150times the clinical UPA exposure are considered significant and supportive to the chronic administration of UPA for more than 3months in women of reproductive age.

Ulipristal acetate versus levonorgestrel-releasing intrauterine system for heavy menstrual bleeding (UCON): a randomised controlled phase III trial.

Background: Heavy menstrual bleeding affects one in four women and negatively impacts quality of life. Ulipristal acetate is prescribed to treat symptoms associated with uterine fibroids. We compared the effectiveness of ulipristal acetate and the levonorgestrel-releasing intrauterine system at reducing the burden of heavy menstrual bleeding, irrespective of the presence of fibroids. Methods: This randomised, open-label, parallel group phase III trial enrolled women over 18 years with heavy menstrual bleeding from 10 UK hospitals. Participants were centrally randomised, in a 1:1 ratio, to either three, 12-week treatment cycles of 5 mg ulipristal acetate daily, separated by 4-week treatment-free intervals, or a levonorgestrel-releasing intrauterine system. The primary outcome, analysed by intention-to-treat, was quality of life measured by the Menorrhagia Multi-Attribute Scale at 12 months. Secondary outcomes included menstrual bleeding and liver function. The trial is registered with ISRCTN, 20426843. Findings: Between June 5th, 2015 and February 26th, 2020, 236 women were randomised, either side of a recruitment suspension due to concerns of ulipristal acetate hepatoxicity. Subsequent withdrawal of ulipristal acetate led to early cessation of recruitment but the trial continued in follow-up. The primary outcome substantially improved in both groups, and was 89, (interquartile range [IQR] 65 to 100, n = 53) and 94, (IQR 70 to 100, n = 50; adjusted odds ratio 0.55, 95% confidence interval [CI] 0.26-1.17; p = 0.12) in the ulipristal and levonorgestrel-releasing intrauterine system groups. Rates of amenorrhoea at 12 months were higher in those allocated ulipristal acetate compared to levonorgestrel-releasing intrauterine system (64% versus 25%, adjusted odds ratio 7.12, 95% CI 2.29-22.2). Other outcomes were similar between the two groups and there were no cases of endometrial malignancy or hepatotoxicity due to ulipristal acetate use. Interpretation: Our findings suggested that both treatments improved quality of life. Ulipristal was more effective at inducing amenorrhoea. Ulipristal has been demonstrated to be an effective medical therapeutic option but currently its use has restrictions and requires liver function monitoring. Funding: UK Medical Research Council and National Institute of Health Research EME Programme (12/206/52).

Endometrial morphology after treatment of uterine fibroids with the selective progesterone receptor modulator, ulipristal acetate.

Selective progesterone receptor modulators (SPRMs) have beneficial effects in reducing the size of uterine fibroids and the amount of bleeding, but their endometrial effects have not been seen with other agents. This report describes the morphology of the endometrium after 3 mo of treatment with the SPRM, ulipristal acetate (UPA). In 2 Phase III randomized double-blind controlled clinical trials, 546 patients with uterine myomas were treated with 5 or 10 mg of UPA daily for 13 wk or placebo or gonadotropin-releasing hormone agonist. Endometrial biopsies were taken at screening, end of treatment (13 wk), and after treatment-free follow-up (38 wk). Biopsies were assessed independently by 3 pathologists according to a preset morphologic scheme. After 13 wk, the UPA-treated endometrium showed altered architectural glandular features including extensive cystic dilatation. The glandular epithelium appeared inactive or contained abortive subnuclear vacuolization, occasional mitoses, and apoptosis. Abnormal stromal vessels were commonly seen. There was a high level of agreement between pathologists on the presence or the absence of nonphysiological changes. One case of hyperplasia without atypia and 4 polyps were seen at 13 wk of UPA treatment. Six months after treatment, the endometrium returned to normal histology in the majority of the patients, with 1 polyp and no cases of hyperplasia in the UPA-treated groups, and 2 hyperplasias (1 with and 1 without atypia) in the placebo or the gonadotropin-releasing hormone-agonist groups. Mild reversible thickening of the endometrium occurs in a minority of cases. It is important that pathologists are aware of the spectrum of changes induced by SPRMs to avoid misdiagnoses of endometrial hyperplasia or polyps.

Improving the Diagnosis of Endometrial Hyperplasia Using Computerized Analysis and Immunohistochemical Biomarkers.

Endometrial hyperplasia (EH) is a precursor lesion to endometrial carcinoma (EC). Risks for EC include genetic, hormonal and metabolic factors most notably those associated with obesity: rates are rising and there is concern that cases in pre-menopausal women may remain undetected. Making an accurate distinction between benign and pre-malignant disease is both a challenge for the pathologist and important to the gynecologist who wants to deliver the most appropriate care to meet the needs of the patient. Premalignant change may be recognized by histological changes of endometrial hyperplasia (which may occur with or without atypia) and endometrial intraepithelial neoplasia (EIN). In this study we created a tissue resource of EH samples diagnosed between 2004 and 2009 (n = 125) and used this to address key questions: 1. Are the EIN/WHO2014 diagnostic criteria able to consistently identify premalignant endometrium? 2. Can computer aided image analysis inform identification of EIN? 3. Can we improve diagnosis by incorporating analysis of protein expression using immunohistochemistry. Our findings confirmed the inclusion of EIN in diagnostic criteria resulted in a better agreement between expert pathologists compared with the previous WHO94 criteria used for the original diagnosis of our sample set. A computer model based on assessment of stromal:epithelial ratio appeared most accurate in classification of areas of tissue without EIN. From an extensive panel of putative endometrial protein tissue biomarkers a score based on assessment of HAND2, PTEN, and PAX2 was able to identify four clusters one of which appeared to be more likely to be benign. In summary, our study has highlighted new opportunities to improve diagnosis of pre-malignant disease in endometrium and provide a platform for further research on this important topic.

Multiomic Characterization of High-Grade Serous Ovarian Carcinoma Enables High-Resolution Patient Stratification.

PURPOSE: High-grade serous ovarian carcinoma (HGSOC) is the most common ovarian cancer type; most patients experience disease recurrence that accumulates chemoresistance, leading to treatment failure. Genomic and transcriptomic features have been associated with differential outcome and treatment response. However, the relationship between events at the gene sequence, copy number, and gene-expression levels remains poorly defined. EXPERIMENTAL DESIGN: We perform multiomic characterization of a large HGSOC cohort (n = 362) with detailed clinical annotation to interrogate the relationship between patient subgroups defined by specific molecular events. RESULTS: BRCA2-mutant (BRCA2m) and EMSY-overexpressing cases demonstrated prolonged survival [multivariable hazard ratios (HR) 0.40 and 0.51] and significantly higher first- and second-line chemotherapy response rate. CCNE1-gained (CCNE1g) cases demonstrated underrepresentation of FIGO stage IV cases, with shorter survival but no significant difference in treatment response. We demonstrate marked overlap between the TCGA- and Tothill-derived subtypes. IMR/C2 cases displayed higher BRCA1/2m frequency (25.5%, 32.5%) and significantly greater immune cell infiltration, whereas PRO/C5 cases had the highest CCNE1g rate (23.9%, 22.2%) and were uniformly low in immune cell infiltration. The survival benefit for cases with aberrations in homologous recombination repair (HRR) genes was apparent across all transcriptomic subtypes (HR range, 0.48-0.68). There was significant co-occurrence of RB loss and HRR gene aberrations; RB loss was further associated with favorable survival within HRR-aberrant cases (multivariable HR, 0.50). CONCLUSIONS: These data paint a high-resolution picture of the molecular landscape in HGSOC, better defining patients who may benefit most from specific molecular therapeutics and highlighting those for whom novel treatment strategies are needed to improve outcomes.

Interleukin-11 in endometrial adenocarcinoma is regulated by prostaglandin F2alpha-F-prostanoid receptor interaction via the calcium-calcineurin-nuclear factor of activated T cells pathway and negatively regulated by the regulator of calcineurin-1.

Interleukin-11 (IL-11) up-regulates the proliferative and invasive capacity of many cancers. Coexpression of glycoprotein 130 (GP130) and IL-11 receptor alpha (IL-11Ralpha) is necessary for high-affinity binding of IL-11 to IL-11Ralpha. This study investigated the expression of IL-11 and role of prostaglandin F(2alpha)-F-prostanoid receptor (FP receptor) signaling in the modulation of IL-11 expression in endometrial adenocarcinoma cells. Localization of IL-11, IL-11Ralpha, and GP130 expression was performed by immunohistochemistry. IL-11 and regulator of calcineurin 1 isoform 4 (RCAN1-4) mRNA and protein expression were determined by real-time RT-PCR and/or enzyme-linked immunosorbent assay/Western blot analysis using Ishikawa endometrial adenocarcinoma cells stably expressing the FP receptor (FPS cells) and endometrial adenocarcinoma explants. IL-11 mRNA expression was significantly elevated in endometrial adenocarcinoma samples compared with normal endometrium and increased with tumor grade. IL-11 protein expression localized with FP receptor, IL-11Ralpha, and GP130 in the neoplastic glandular epithelium of endometrial adenocarcinomas. Prostaglandin F(2alpha)-FP receptor signaling significantly elevated the expression of IL-11 mRNA and protein in a Gq-protein kinase C-calcium-calcineurin-nuclear factor of activated T cells-dependent manner in FPS cells. The calcineurin signaling pathway is known to be controlled by the RCAN (RCAN1-4). Indeed, RCAN1-4 expression was significantly elevated in well-differentiated endometrial adenocarcinoma compared with normal endometrium and was found to decrease with tumor grade and negatively regulate IL-11 expression in vitro. This study has highlighted a new mechanism regulating IL-11 expression in endometrial adenocarcinoma cells by the FP receptor via the calcium-calcineurin-nuclear factor of activated T cells pathway.

BRCA1 is both a prognostic and predictive biomarker of response to chemotherapy in sporadic epithelial ovarian cancer.

OBJECTIVES: We investigated the relationship between BRCA1 protein expression by immunohistochemistry (IHC) and clinical outcome following platinum and platinum/taxane chemotherapy in sporadic epithelial ovarian cancer (EOC). METHODS: BRCA1 IHC was performed on a cohort of 292 ovarian tumours from two UK oncology centres. BRCA1 protein expression levels were correlated with overall survival (OS), progression free survival (PFS) and clinical response to chemotherapy by multivariate analysis. RESULTS: EOC patients with absent/low BRCA1 protein expression (41%) had a better chance of clinical response following chemotherapy as compared to patients with high BRCA1 expression (odds ratio 2.47: 95%CI 1.10-5.55, p=0.029). Patients with absent/low BRCA1 had a higher probability of clinical response following single agent platinum compared to high BRCA1 expressing patients (68.5% vs. 46.8%), while addition of a taxane increased response rates independent of BRCA1. Overall, patients with absent/low BRCA1 had a better clinical outcome compared to patients with high BRCA1 protein expression in terms of both OS (HR=0.65: 95%CI 0.48-0.88, p=0.006) and PFS (HR=0.74, 95%CI 0.55-0.98, p=0.040). CONCLUSIONS: We confirm that absent/low BRCA1 protein expression is a favourable prognostic marker. However, we also provide the first evidence that absent/low BRCA1 protein expression in sporadic EOC patients predicts for an improved clinical response to chemotherapy.

The endometrial response to modulation of ligand-progesterone receptor pathways is reversible.

OBJECTIVE: To study the impact of the progesterone receptor modulator (PRM), ulipristal acetate (UPA), on endometrial morphology and function. DESIGN: Cross-sectional. SETTING: University Research Institute. PATIENT(S): Endometrial biopsies from 16 patients with heavy menstrual bleeding with a structurally normal uterus or in association with structural abnormalities identified on radiological imaging (fibroids, adenomyosis or a combination of fibroids and adenomyosis). INTERVENTION(S): Participants received UPA (5 mg once daily) for three 12-week courses, each separated by 4 weeks without treatment. MAIN OUTCOME MEASURE(S): Gene expression by real-time quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and digital image analysis were analyzed to investigate the endometrial impact of modulation of progesterone receptor pathways upon expression of steroid receptors, steroid metabolizing enzymes, cell proliferation, and progesterone-regulated genes in the same patients at 3 time points: before, during, and after discontinuation of PRM treatment. RESULT(S): Ulipristal acetate treatment resulted in increased messenger ribonucleic acid (mRNA) levels of steroid receptors compared with pretreatment secretory endometrium; decreased mRNA levels of 17- and 11-beta-hydroxysteroid dehydrogenases compared with pretreatment proliferative endometrium and pretreatment secretory endometrium; reduced cell proliferation compared with pretreatment proliferative endometrium; and altered mRNA levels of progesterone-regulated genes. A strong consistency between immunohistochemistry-digital image analysis and real-time quantitative reverse transcription polymerase chain reaction results was evident. Alterations in the mRNA levels and endometrial morphology returned to a pretreatment phenotype after the cessation of PRM exposure. CONCLUSION(S): The endometrial impact of the modulation of progesterone receptor pathways with PRM (UPA) treatment is reversible. CLINICAL TRIAL REGISTRATION NUMBER: Ulipristal acetate versus conventional management of heavy menstrual bleeding (UCON) trial (EudraCT 2014-003408-65; REC14/LO/1602).

Prostaglandin F(2alpha)-F-prostanoid receptor regulates CXCL8 expression in endometrial adenocarcinoma cells via the calcium-calcineurin-NFAT pathway.

Pro-inflammatory mediators, like prostaglandin (PG) and chemokines, promote tumourigenesis by enhancing cell proliferation, migration of immune cells and recruitment of blood vessels. Recently we showed elevated expression of the chemokine (C-X-C motif) receptor 2 (CXCR2) in endometrial adenocarcinomas localized to neutrophils and neoplastic epithelial and vascular cells. Furthermore we found that PGF(2alpha)-F-prostanoid (FP) receptor regulates the expression of the CXCR2 ligand CXCL1, to promote neutrophil chemotaxis in endometrial adenocarcinomas. In the present study we identified another CXCR2 ligand, CXCL8 as a target for PGF(2alpha)-FP receptor signalling which enhances epithelial cell proliferation in endometrial adenocarcinoma cells in vitro and in nude mice in vivo. We found that PGF(2alpha)-FP receptor interaction induces CXCL8 expression in endometrial adenocarcinoma cells via the protein kinase C-calcium-calcineurin-NFAT signaling pathway. Promoter analysis revealed that CXCL8 transcriptional activation by PGF(2alpha) signaling is mediated by cooperative interactions between the AP1 and NFAT binding sites. Furthermore, PGF(2alpha) via the FP receptor induced the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway in a reciprocal manner to CXCL8. Using an adenovirus to overexpress RCAN1-4, we found that RCAN1-4 is a negative regulator of CXCL8 expression in endometrial adenocarcinoma cells. Taken together our data have elucidated the molecular and cellular mechanism whereby PGF(2alpha) regulates CXCL8 expression via the FP receptor in endometrial adenocarcinomas and have highlighted RCAN1-4 as a negative regulator of CXCL8 expression which may be exploited therapeutically to inhibit CXCL8-mediated tumour development.

Effects of the levonorgestrel intrauterine system on the endometrium after long-term exposure to mifepristone: Secondary outcomes of a randomized controlled trial.

OBJECTIVE: Long-term treatment with progesterone receptor modulators (PRM) is associated with a distinct histological entity termed progesterone receptor modulator associated endometrial changes (PAEC). While accumulating evidence implies that these changes are benign and reversible after cessation of treatment, there are currently no data underpinning their development. Consequently, as a precaution, endometrial shedding is recommended after long-term PRM intake. Avoiding endometrial shedding after treatment with a PRM and prior to the start of a progestin treatment would be beneficial for women in reproductive age to avoid pregnancy and bleeding. However, the endometrial morphology with such a treatment regimen is unknown. The aim of this study was to delineate the endometrial morphology following continuous long-term treatment with the PRM mifepristone and subsequent placement of a levonorgestrel intrauterine system (LNG-IUS) without prior shedding of the endometrium. STUDY DESIGN: This study reports the secondary outcome from a double-blinded randomized controlled trial conducted at Karolinska University Hospital, Sweden, November 2009 to January 2015. Healthy women aged 18-43 years with regular menstrual cycles were included. Eligible women were randomized to receive either 50 mg of mifepristone (n = 29) or a comparator (n = 29), every other day for two months followed by insertion of an LNG-IUS 52 mg. Endometrial biopsies were obtained at baseline and three months after placement of the device. The samples were histologically assessed. The main outcome measure of this sub-study was the endometrial morphology including presence of PAEC three months after LNG-IUS insertion. RESULTS: Nine and eight paired biopsies from the mifepristone and comparator group, respectively, were included in the histological analysis. There were no differences in baseline characteristics between the groups and all baseline endometrial biopsies were physiological. Three months after LNG-IUS placement the endometrial morphology was still benign without PAEC in all samples treated with either mifepristone or comparator. A progestin effect on the endometrium was seen in all samples. CONCLUSIONS: Placement of an LNG-IUS immediately following two months' treatment with the PRM mifepristone, without any prior shedding of the endometrium, may represent a feasible approach in terms of endometrial safety. However, larger studies are needed to confirm our results.

Expression of oestrogen receptors, ERalpha, ERbeta, and ERbeta variants, in endometrial cancers and evidence that prostaglandin F may play a role in regulating expression of ERalpha.

BACKGROUND: Endometrial cancer is the most common gynaecological malignancy; risk factors include exposure to oestrogens and high body mass index. Expression of enzymes involved in biosynthesis of oestrogens and prostaglandins (PG) is often higher in endometrial cancers when compared with levels detected in normal endometrium. Oestrogens bind one of two receptors (ERalpha and ERbeta) encoded by separate genes. The full-length receptors function as ligand-activated transcription factors; splice variant isoforms of ERbeta lacking a ligand-binding domain have also been described. PGs act in an autocrine or paracrine manner by binding to specific G-protein coupled receptors. METHODS: We compared expression of ERs, progesterone receptor (PR) and cyclooxygenase-2 (COX-2) in stage 1 endometrial adenocarcinomas graded as well (G1), moderately (G2) or poorly (G3) differentiated (n >or= 10 each group) using qRTPCR, single and double immunohistochemistry. We used endometrial adenocarcinoma cell lines to investigate the impact of PGF2alpha on expression of ERs and PR. RESULTS: Full length ERbeta (ERbeta1) and two ERbeta variants (ERbeta2, ERbeta5) were expressed in endometrial cancers regardless of grade and the proteins were immunolocalised to the nuclei of cells in both epithelial and stromal compartments. Immunoexpression of COX-2 was most intense in cells that were ERalphaneg/low. Expression of PR in endometrial adenocarcinoma (Ishikawa) cell lines and tissues broadly paralleled that of ERalpha. Treatment of adenocarcinoma cells with PGF2alpha reduced expression of ERalpha but had no impact on ERbeta1. Cells incubated with PGF2alpha were unable to increase expression of PR mRNA when they were incubated with E2. CONCLUSION: We have demonstrated that ERbeta5 protein is expressed in stage 1 endometrial adenocarcinomas. Expression of three ERbeta variants, including the full-length protein is not grade-dependent and most cells in poorly differentiated cancers are ERbetapos/ERalphaneg. We found evidence of a link between COX-2, its product PGF2alpha, and expression of ERalpha and PR that sheds new light on the cross talk between steroid and PG signalling pathways in this disease.

Development of an in-house ELISA to detect anti-HPV16-L1 antibodies in serum and dried blood spots.

Measuring anti-HPV antibody levels is important for surveillance of the immunological response to both natural infection and vaccination. Here, an ELISA test for measurement of HPV-16L1 antibodies was developed and validated in sera and dried blood spots. An in-house ELISA was developed for measuring anti-HPV-16L1 IgA and IgG levels. The assay was standardized against WHO international standard serum and validated on serum, dried blood spots and cervical liquid based cytology samples from women attending colposcopy clinics in Scotland. Antibody avidity index was also measured in serum samples. The average HPV 16-L1 specific IgG and IgA levels measured in sera, in women attending a routine colposcopy service were 7.3 units/ml and 8.1 units/ml respectively. Significant correlations between serum and dried blood spot eluates for both IgG and IgA were observed indicating that the latter serve as a credible proxy for antibody levels. Average IgG Avidity Index was 35% (95% CI 25%-45%) suggesting previous, historical challenge with natural infection. This ELISA has potential for use in epidemiological and field studies of antibody prevalence and if coupled with avidity measurement may be of use in individual case monitoring of vaccine responses and failures.

Effects of the selective progesterone receptor modulator asoprisnil on uterine artery blood flow, ovarian activity, and clinical symptoms in patients with uterine leiomyomata scheduled for hysterectomy.

INTRODUCTION: Asoprisnil, a novel orally active selective progesterone receptor modulator, is being studied for the management of symptomatic uterine leiomyomata. The exact mechanism of action is not yet discerned. The primary objectives of this double-blind, randomized, placebo-controlled study included evaluation of the effect of asoprisnil on uterine artery blood flow. Furthermore, we assessed effects of asoprisnil on leiomyoma symptoms. PATIENTS AND METHODS: Thirty-three premenopausal patients scheduled for hysterectomy due to symptomatic uterine leiomyomata were recruited in four centers and treated with 10 or 25 mg asoprisnil or placebo for 12 wk before surgery. At baseline and before hysterectomy, all patients underwent sonographic assessment to measure impedance to uterine artery blood flow, determined by resistance index and pulsatility index, as well as volumes of largest leiomyoma and uterus. In addition, patients recorded intensity and frequency of menstrual bleeding on a menstrual pictogram. Each asoprisnil treatment was compared with placebo. RESULTS: The increased pulsatility index in both asoprisnil groups and the statistically significantly increased resistance index within the 25-mg asoprisnil group suggest a moderately decreased uterine artery blood flow. Analysis of menstrual pictogram scores showed a statistically significant larger decrease in frequency and intensity of bleeding for both asoprisnil groups compared with placebo. Bleeding was suppressed by asoprisnil 25mg in 91% of patients. Asoprisnil treatment was well tolerated when administered daily for a 12-wk period, and no serious adverse events occurred. CONCLUSION: Asoprisnil moderately reduced uterine artery blood flow. This effect may contribute in part to the clinical effects of asoprisnil.

Insulin-like growth factor binding proteins IGFBP3, IGFBP4, and IGFBP5 predict endocrine responsiveness in patients with ovarian cancer.

PURPOSE: This study sought to explore the predictive value of the insulin-like growth factor (IGF) binding proteins (IGFBP) as markers of response in ovarian cancer patients treated with the aromatase inhibitor letrozole. EXPERIMENTAL DESIGN: IGFBP mRNA expression in cell lines was measured by quantitative reverse transcription-PCR and IGFBP protein expression measured in sections from primary tumors of patients treated with letrozole by semiquantitative immunohistochemistry. RESULTS: Quantitative reverse transcription-PCR analysis showed that IGFBP3 and IGFBP5 were down-regulated and IGFBP4 was up-regulated by 17beta-estradiol (E(2)) in an estrogen receptor (ER)-positive ovarian cancer cell line. Expressions of IGFBP1, IGFBP2, and IGFBP6 were unaffected by E(2). The E(2) modulation of these genes was reversed by tamoxifen. Using ERalpha-specific (propyl pyrazole triol) and ERbeta-specific (diarylpropionitrile) agonists, the gene expression modulations produced by E(2) could be replicated by propyl pyrazole triol but not by diarylpropionitrile. For ovarian cancer patients being treated with letrozole, we tested the predictive value of the IGFBPs in paraffin-fixed sections from their primary tumors by semiquantitative immunohistochemistry. Using serum CA125 as an indicator of progression/response, significant differences in expression levels of IGFBPs were observed between tumors from CA125 responding/stable patients compared with tumors from progressing patients. Mean immunoscores for IGFBP3 and IGFBP5 were significantly lower, and mean expression of IGFBP4 was significantly higher in tumors from patients demonstrating CA125 response or stabilization compared with CA125 progression. CONCLUSION: These results indicate that expression levels of certain IGFBP family members in ovarian cancers are estrogen regulated and can, thus, help identify patients who could benefit from endocrine therapy.