Menopausal Hormone Therapy and the Breast: A Review of Clinical Studies.

Background: Women in the peri- or postmenopause can experience symptoms related to the gradual degradation of ovarian function. Hormone replacement therapy (HRT) is the most effective therapy to treat common menopausal symptoms such as hot flashes and vaginal discomfort. However, safety concerns have been raised revolving, among others also, around the risk of breast cancer. Methods: This article is based on a selective literature search for relevant studies regarding HRT use and the risk of breast cancer in the general population or BRCA carriers, the risk of breast cancer recurrence, or the risk of breast cancer in situ. Summary: HRT can lead to little or no increase in breast cancer risk. The risk depends on the duration and composition of the HRT and decreases after stopping the treatment. Data assessing the oncological safety of HRT after breast cancer are inconsistent. According to current knowledge, HRT is fundamentally contraindicated after breast cancer but can be individually considered after a risk-benefit assessment and when nonhormonal therapies have failed. The same applies to HRT after DCIS, which should not be routinely offered but nonetheless can be considered in individual cases. HRT can be offered up to the age of natural menopause for BRCA mutation carriers who are undergoing risk-reducing bilateral salpingo-oophorectomy and do not have a personal history of breast cancer, but is contraindicated in BRCA mutation carriers who have already had breast cancer.

Endometrial Cancer. Guideline of the DGGG, DKG and DKH (S3-Level, AWMF Registry Number 032/034-OL, September 2022). Part 1 with Recommendations on the Epidemiology, Screening, Diagnosis and Hereditary Factors of Endometrial Cancer, Geriatric Assessment and Supply Structures.

Summary The S3-guideline on endometrial cancer, first published in April 2018, was reviewed in its entirety between April 2020 and January 2022 and updated. The review was carried out at the request of German Cancer Aid as part of the Oncology Guidelines Program and the lead coordinators were the German Society for Gynecology and Obstetrics (DGGG), the Gynecology Oncology Working Group (AGO) of the German Cancer Society (DKG) and the German Cancer Aid (DKH). The guideline update was based on a systematic search and assessment of the literature published between 2016 and 2020. All statements, recommendations and background texts were reviewed and either confirmed or amended. New statements and recommendations were included where necessary. Aim The use of evidence-based risk-adapted therapies to treat women with endometrial cancer of low risk prevents unnecessarily radical surgery and avoids non-beneficial adjuvant radiation therapy and/or chemotherapy. For women with endometrial cancer and a high risk of recurrence, the guideline defines the optimum level of radical surgery and indicates whether chemotherapy and/or adjuvant radiation therapy is necessary. This should improve the survival rates and quality of life of these patients. The S3-guideline on endometrial cancer and the quality indicators based on the guideline aim to provide the basis for the work of certified gynecological cancer centers. Methods The guideline was first compiled in 2018 in accordance with the requirements for S3-level guidelines and was updated in 2022. The update included an adaptation of the source guidelines identified using the German Instrument for Methodological Guideline Appraisal (DELBI). The update also used evidence reviews which were created based on selected literature obtained from systematic searches in selected literature databases using the PICO process. The Clinical Guidelines Service Group was tasked with carrying out a systematic search and assessment of the literature. Their results were used by interdisciplinary working groups as a basis for developing suggestions for recommendations and statements which were then modified during structured online consensus conferences and/or additionally amended online using the DELPHI process to achieve a consensus. Recommendations Part 1 of this short version of the guideline provides recommendations on epidemiology, screening, diagnosis, and hereditary factors. The epidemiology of endometrial cancer and the risk factors for developing endometrial cancer are presented. The options for screening and the methods used to diagnose endometrial cancer are outlined. Recommendations are given for the prevention, diagnosis, and therapy of hereditary forms of endometrial cancer. The use of geriatric assessment is considered and existing structures of care are presented.

Phytoestrogen Treatment for Menopausal Vasomotor Symptoms after Breast Cancer.

Introduction: In breast cancer patients, menopausal symptoms are often reached earlier and in a more severe manner than in healthy women. They can dramatically reduce a patient's quality of life. Treatment for breast cancer patients remains difficult due to hormone replacement therapy being contraindicated. Therefore, treatment alternatives like herbal phytoestrogen preparations are considered to be a treatment option. Methods: All randomized controlled trials (RCTs) that investigated comparisons of food products, extracts, or dietary supplements containing phytoestrogens in women with a diagnosis of breast cancer were included in the analysis. Results: There was no evidence that phytoestrogen preparations had a benefit over placebo in the treatment of breast cancer patients with menopausal symptoms. Neither the frequency nor severity of menopausal symptoms and the quality of life or occurrence of adverse events were reduced in comparison to a placebo. Conclusion: As we cannot prove a benefit of plant extracts in the treatment of menopausal symptoms in breast cancer patients, different herbal preparations should be analyzed in RCTs in order to find sufficient and safe new treatment options for symptomatic menopausal breast cancer patients. A focus should also be laid on developing a core outcome set to simplify pooling of different studies.

Role of Syndecans in Ovarian Cancer: New Diagnostic and Prognostic Biomarkers and Potential Therapeutic Targets.

Ovarian cancer (OC) is the eighth cancer both in prevalence and mortality in women and represents the deadliest female reproductive cancer. Due to generally vague symptoms, OC is frequently diagnosed only at a late and advanced stage, resulting in high mortality. The tumor extracellular matrix and cellular matrix receptors play a key role in the pathogenesis of tumor progression. Syndecans are a family of four transmembrane heparan sulfate proteoglycans (PG), including syndecan-1, -2, -3, and -4, which are dysregulated in a myriad of cancers, including OC. Many clinicopathological studies suggest that these proteins are promising diagnostic and prognostic biomarkers for OC. Furthermore, functions of the syndecan family in the regulation of cellular processes make it an interesting pharmacological target for anticancer therapies.

Progesterone from ovulatory menstrual cycles is an important cause of breast cancer.

Many factors, including reproductive hormones, have been linked to a woman's risk of developing breast cancer (BC). We reviewed the literature regarding the relationship between ovulatory menstrual cycles (MCs) and BC risk. Physiological variations in the frequency of MCs and interference with MCs through genetic variations, pathological conditions and or pharmaceutical interventions revealed a strong link between BC risk and the lifetime number of MCs. A substantial reduction in BC risk is observed in situations without MCs. In genetic or transgender situations with normal female breasts and estrogens, but no progesterone (P4), the incidence of BC is very low, suggesting an essential role of P4. During the MC, P4 has a strong proliferative effect on normal breast epithelium, whereas estradiol (E2) has only a minimal effect. The origin of BC has been strongly linked to proliferation associated DNA replication errors, and the repeated stimulation of the breast epithelium by P4 with each MC is likely to impact the epithelial mutational burden. Long-lived cells, such as stem cells, present in the breast epithelium, can carry mutations forward for an extended period of time, and studies show that breast tumors tend to take decades to develop before detection. We therefore postulate that P4 is an important factor in a woman's lifetime risk of developing BC, and that breast tumors arising during hormonal contraception or after menopause, with or without menopausal hormone therapy, are the consequence of the outgrowth of pre-existing neoplastic lesions, eventually stimulated by estrogens and some progestins.

Tumor suppressor miR-218 directly targets epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer, sensitizing cells to irradiation.

PURPOSE: MicroRNA-218 (miR-218) is a key regulator of numerous processes relevant to tumor progression. In the present study, we aimed to characterize the relationship between miR-218 and the Epidermal Growth Factor Receptor (EGFR) as well as to understand downstream effects in triple-negative breast cancer (TNBC). METHODS: We assessed miR-218 and EGFR expression in cell lines and publicly available primary breast cancer gene expression data. We then overexpressed miR-218 in two TNBC cell lines and investigated effects on EGFR and downstream mitogen-activated protein (MAP) kinase signaling. Luciferase reporter assay was used to characterize a direct binding interaction between miR-218 and EGFR mRNA. Digital holographic microscopy helped investigate cell migration and dry mass after miR-218 overexpression. Cell division and invasion were assessed microscopically, while radiation response after miR-218 overexpression alone or combined with additional EGFR knockdown was investigated via clonogenic assays. RESULTS: We found an inverse correlation between EGFR expression and miR-218 levels in cell lines and primary breast cancer tissues. MiR-218 overexpression resulted in a downregulation of EGFR via direct binding of the mRNA. Activation of EGFR and downstream p44/42 MAPK signaling were reduced after pre-miR-218 transfection. Cell proliferation, motility and invasiveness were inhibited whereas cell death and mitotic catastrophe were upregulated in miR-218 overexpressing cells compared to controls. MiR-218 overexpressing and EGFR siRNA-treated cells were sensitized to irradiation, more than miR-218 overexpressing cells alone. CONCLUSION: This study characterizes the antagonistic relationship between miR-218 and EGFR. It also demonstrates downstream functional effects of miR-218 overexpression, leading to anti-tumorigenic cellular changes.

The Tissue Factor Pathway in Cancer: Overview and Role of Heparan Sulfate Proteoglycans.

Historically, the only focus on tissue factor (TF) in clinical pathophysiology has been on its function as the initiation of the extrinsic coagulation cascade. This obsolete vessel-wall TF dogma is now being challenged by the findings that TF circulates throughout the body as a soluble form, a cell-associated protein, and a binding microparticle. Furthermore, it has been observed that TF is expressed by various cell types, including T-lymphocytes and platelets, and that certain pathological situations, such as chronic and acute inflammatory states, and cancer, may increase its expression and activity. Transmembrane G protein-coupled protease-activated receptors can be proteolytically cleaved by the TF:FVIIa complex that develops when TF binds to Factor VII (PARs). The TF:FVIIa complex can activate integrins, receptor tyrosine kinases (RTKs), and PARs in addition to PARs. Cancer cells use these signaling pathways to promote cell division, angiogenesis, metastasis, and the maintenance of cancer stem-like cells. Proteoglycans play a crucial role in the biochemical and mechanical properties of the cellular extracellular matrix, where they control cellular behavior via interacting with transmembrane receptors. For TFPI.fXa complexes, heparan sulfate proteoglycans (HSPGs) may serve as the primary receptor for uptake and degradation. The regulation of TF expression, TF signaling mechanisms, their pathogenic effects, and their therapeutic targeting in cancer are all covered in detail here.

Establishment of a 3D co-culture model to investigate the role of primary fibroblasts in ductal carcinoma in situ of the breast.

BACKGROUND: Ductal carcinoma in situ (DCIS) is a precursor form of breast cancer. 13%-50% of these lesions will progress to invasive breast cancer, but the individual progression risk cannot be estimated. Therefore, all patients receive the same therapy, resulting in potential overtreatment of a large proportion of patients. AIMS: The role of the tumor microenvironment (TME) and especially of fibroblasts appears to be critical in DCIS development and a better understanding of their role may aid individualized treatment. METHODS AND RESULTS: Primary fibroblasts isolated from benign or malignant punch biopsies of the breast and MCF10DCIS.com cells were seeded in a 3D cell culture system. The fibroblasts were cultured in a type I collagen layer beneath a Matrigel layer with MCF10DCIS.com cells. Dye-quenched (DQ) fluorescent collagen I and IV were used in collagen and Matrigel layer respectively to demonstrate proteolysis. Confocal microscopy was performed on day 2, 7, and 14 to reveal morphological changes, which could indicate the transition to an invasive phenotype. MCF10DCIS.com cells form smooth, round spheroids in co-culture with non-cancer associated fibroblasts (NAFs). Spheroids in co-culture with tumor-associated fibroblasts (TAFs) appear irregularly shaped and with an uneven surface; similar to spheroids formed from invasive cells. Therefore, these morphological changes represent the progression of an in situ to an invasive phenotype. In addition, TAFs show a higher proteolytic activity compared to NAFs. The distance between DCIS cells and fibroblasts decreases over time. CONCLUSION: The TAFs seem to play an important role in the progression of DCIS to invasive breast cancer. The better characterization of the TME could lead to the identification of DCIS lesions with high or low risk of progression. This could enable personalized oncological therapy, prevention of overtreatment and individualized hormone replacement therapy after DCIS.

Anti-Hormonal Therapy in Breast Cancer and Its Effect on the Blood-Brain Barrier.

The molecular receptor status of breast cancer has implications for prognosis and long-term metastasis. Although metastatic luminal B-like, hormone-receptor-positive, HER2-negative, breast cancer causes brain metastases less frequently than other subtypes, though tumor metastases in the brain are increasingly being detected of this patient group. Despite the many years of tried and tested use of a wide variety of anti-hormonal therapeutic agents, there is insufficient data on their intracerebral effectiveness and their ability to cross the blood-brain barrier. In this review, we therefore summarize the current state of knowledge on anti-hormonal therapy and its intracerebral impact and effects on the blood-brain barrier in breast cancer.

Aromatase inhibitors (letrozole) for ovulation induction in infertile women with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common cause of infrequent periods (oligomenorrhoea) and absence of periods (amenorrhoea). It affects about 5% to 20% of women worldwide and often leads to anovulatory infertility. Aromatase inhibitors (AIs) are a class of drugs that were introduced for ovulation induction in 2001. Since about 2001 clinical trials have reached differing conclusions as to whether the AI, letrozole, is at least as effective as the first-line treatment clomiphene citrate (CC), a selective oestrogen receptor modulator (SERM). OBJECTIVES: To evaluate the effectiveness and safety of AIs (letrozole) (with or without adjuncts) compared to SERMs (with or without adjuncts) for infertile women with anovulatory PCOS for ovulation induction followed by timed intercourse or intrauterine insemination. SEARCH METHODS: We searched the following sources, from their inception to 4 November 2021, to identify relevant randomised controlled trials (RCTs): the Cochrane Gynaecology and Fertility Group Specialised Register, CENTRAL, MEDLINE, Embase and PsycINFO. We also checked reference lists of relevant trials, searched the trial registers and contacted experts in the field for any additional trials. We did not restrict the searches by language or publication status. SELECTION CRITERIA: We included all RCTs of AIs used alone or with other medical therapies for ovulation induction in women of reproductive age with anovulatory PCOS. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials, extracted the data and assessed risks of bias using RoB 1. We pooled trials where appropriate using a fixed-effect model to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for most outcomes, and risk differences (RDs) for ovarian hyperstimulation syndrome (OHSS). The primary outcomes were live birth rate and OHSS rate. Secondary outcomes were clinical pregnancy, miscarriage and multiple pregnancy rates. We assessed the certainty of the evidence for each comparison using GRADE methods. MAIN RESULTS: This is a substantive update of a previous review; of six previously included trials, we excluded four from this update and moved two to 'awaiting classification' due to concerns about validity of trial data. We included five additional trials for this update that now includes a total of 41 RCTs (6522 women). The AI, letrozole, was used in all trials. Letrozole compared to SERMs with or without adjuncts followed by timed intercourse Live birth rates were higher with letrozole (with or without adjuncts) compared to SERMs followed by timed intercourse (OR 1.72, 95% CI 1.40 to 2.11; I2 = 0%; number needed to treat for an additional beneficial outcome (NNTB) = 10; 11 trials, 2060 participants; high-certainty evidence). This suggests that in women with a 20% chance of live birth using SERMs, the live birth rate in women using letrozole with or without adjuncts would be 27% to 35%. There is high-certainty evidence that OHSS rates are similar with letrozole or SERMs (0.5% in both arms: risk difference (RD) -0.00, 95% CI -0.01 to 0.01; I2 = 0%; 10 trials, 1848 participants; high-certainty evidence). There is evidence for a higher pregnancy rate in favour of letrozole (OR 1.69, 95% CI 1.45 to 1.98; I2 = 0%; NNTB = 10; 23 trials, 3321 participants; high-certainty evidence). This suggests that in women with a 24% chance of clinical pregnancy using SERMs, the clinical pregnancy rate in women using letrozole with or without adjuncts would be 32% to 39%. There is little or no difference between treatment groups in the rate of miscarriage per pregnancy (25% with SERMs versus 24% with letrozole: OR 0.94, 95% CI 0.66 to 1.32; I2 = 0%; 15 trials, 736 participants; high-certainty evidence) and multiple pregnancy rate (2.2% with SERMs versus 1.6% with letrozole: OR 0.74, 95% CI 0.42 to 1.32; I2 = 0%; 14 trials, 2247 participants; high-certainty evidence). However, a funnel plot showed mild asymmetry, indicating that some trials in favour of SERMs might be missing.  Letrozole compared to laparoscopic ovarian drilling (LOD) One trial reported very low-certainty evidence that live birth rates may be higher with letrozole compared to LOD (OR 2.07, 95% CI 0.99 to 4.32; 1 trial, 141 participants; very low-certainty evidence). This suggests that in women with a 22% chance of live birth using LOD with or without adjuncts, the live birth rate in women using letrozole with or without adjuncts would be 24% to 47%. No trial reported OHSS rates. Due to the low-certainty evidence we are uncertain if letrozole improves pregnancy rates compared to LOD (OR 1.47, 95% CI 0.95 to 2.28; I² = 0%; 3 trials, 367 participants; low-certainty evidence). This suggests that in women with a 29% chance of clinical pregnancy using LOD with or without adjuncts, the clinical pregnancy rate in women using letrozole with or without adjuncts would be 28% to 45%. There seems to be no evidence of a difference in miscarriage rates per pregnancy comparing letrozole to LOD (OR 0.65, 95% CI 0.22 to 1.92; I² = 0%; 3 trials, 122 participants; low-certainty evidence). This also applies to multiple pregnancies (OR 3.00, 95% CI 0.12 to 74.90; 1 trial, 141 participants; very low-certainty evidence). AUTHORS' CONCLUSIONS: Letrozole appears to improve live birth rates and pregnancy rates in infertile women with anovulatory PCOS, compared to SERMs, when used for ovulation induction, followed by intercourse. There is high-certainty evidence that OHSS rates are similar with letrozole or SERMs. There was high-certainty evidence of no difference in miscarriage rate and multiple pregnancy rate. We are uncertain if letrozole increases live birth rates compared to LOD. In this update, we added good quality trials and removed trials with concerns over data validity, thereby upgrading the certainty of the evidence base.

The hyaluronan-related genes HAS2, HYAL1-4, PH20 and HYALP1 are associated with prognosis, cell viability and spheroid formation capacity in ovarian cancer.

PURPOSE: Hyaluronan modulates tumour progression, including cell adhesion, cohesion, proliferation and invasion, and the cancer stem cell phenotype. In ovarian cancer, high levels of stromal hyaluronan are associated with poor prognosis. In this work, hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-4, PH-20, HYALP1) were examined with regard to different levels of gene expression and its influence on ovarian cancer patients' survival. The impact of a siRNA depletion of HAS2 was investigated in vitro. METHODS: Using the Kaplan-Meier Plotter tool, we investigated the influence of hyaluronic synthases and hyaluronidases on the survival of a collective of 1435 ovarian cancer patients. Differences in gene expression between normal (n = 46) and cancerous (n = 744) ovarian tissue were examined using the TNMplot database. Following an evaluation of hyaluronan-related gene expression in the ATCC ovarian cancer panel, we studied SKOV3 and SW 626 ovarian cancer cells subjected to HAS2 siRNA or control siRNA treatment in terms of HAS1-3, HYAL2 and HYAL3 mRNA expression. We investigated the ability to form spheroids using the Hanging Drop method and the response to chemotherapy at different concentrations using the MTT Assay. By STRING analysis, interactions within the enzymes of the hyaluronic acid system and with binding partners were visualized. RESULTS: HAS1, HYAL1 and HYAL4 mRNA expression is significantly upregulated, whereas HAS2, HYAL2 and HYAL3 mRNA expression is significantly downregulated in ovarian cancer tissue compared to controls. HAS2 improves cell viability, the capability to form tumour spheroids and has a negative prognostic value regarding overall survival. Lower HAS2 expression and high expression of HYAL2 and HYAL3 favours the survival of ovarian cancer patients. HAS2 knockdown cells and control cells showed a moderate response to combinatorial in vitro chemotherapy with taxol and cisplatin. CONCLUSION: In conclusion, our study shows that the hyaluronic acid system has a relevant influence on the survival of ovarian cancer patients and could therefore be considered as a possible prognostic factor.

The Cell Surface Heparan Sulfate Proteoglycan Syndecan-3 Promotes Ovarian Cancer Pathogenesis.

Syndecans are transmembrane heparan sulfate proteoglycans that integrate signaling at the cell surface. By interacting with cytokines, signaling receptors, proteases, and extracellular matrix proteins, syndecans regulate cell proliferation, metastasis, angiogenesis, and inflammation. We analyzed public gene expression datasets to evaluate the dysregulation and potential prognostic impact of Syndecan-3 in ovarian cancer. Moreover, we performed functional in vitro analysis in syndecan-3-siRNA-treated SKOV3 and CAOV3 ovarian cancer cells. In silico analysis of public gene array datasets revealed that syndecan-3 mRNA expression was significantly increased 5.8-fold in ovarian cancer tissues (n = 744) and 3.4-fold in metastases (n = 44) compared with control tissue (n = 46), as independently confirmed in an RNAseq dataset on ovarian serous cystadenocarcinoma tissue (n = 374, controls: n = 133, 3.5-fold increase tumor vs. normal). Syndecan-3 siRNA knockdown impaired 3D spheroid growth and colony formation as stemness-related readouts in SKOV3 and CAOV3 cells. In SKOV3, but not in CAOV3 cells, syndecan-3 depletion reduced cell viability both under basal conditions and under chemotherapy with cisplatin, or cisplatin and paclitaxel. While analysis of the SIOVDB database did not reveal differences in Syndecan-3 expression between patients, sensitive, resistant or refractory to chemotherapy, KM Plotter analysis of 1435 ovarian cancer patients revealed that high syndecan-3 expression was associated with reduced survival in patients treated with taxol and platin. At the molecular level, a reduction in Stat3 activation and changes in the expression of Wnt and notch signaling constituents were observed. Our study suggests that up-regulation of syndecan-3 promotes the pathogenesis of ovarian cancer by modulating stemness-associated pathways.

The heparan sulphate proteoglycan Syndecan-1 (CD138) regulates tumour progression in a 3D model of ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) is a form of breast cancer that is restricted to the lactiferous ducts and has not yet invaded the surrounding breast tissue. Dysregulation of the transmembrane heparan sulphate proteoglycan Syndecan-1 (Sdc-1) plays a role in tumour progression of invasive breast cancer (IBC). In DCIS, Sdc-1, c-Met and E-cadherin are part of a proangiogenic expression signature. In this study, we employed a siRNA knockdown approach in the DCIS model cell line MCF10A DCIS.com to investigate a potential connection between Sdc-1 and epithelial mesenchymal transition (EMT), proteolysis and the Rho kinase pathway. Analysis of gene expression data of the TNMplot.com database revealed that Sdc-1 expression was higher in primary breast tumours compared to metastases. The impact of Sdc-1-depletion on the cellular phenotype was investigated in a Matrigel-based three-dimensional cell culture model. Sdc-1 depletion resulted in the formation of larger spheroids and the formation of invasive protrusions. Application of matrix metalloproteinase (MMP) and Rho kinase inhibitors could block the Sdc-1-induced phenotype. qPCR analysis of Sdc-1-depleted cells in two-dimensional culture revealed upregulated expression of the EMT-markers CDH1, FN-1, CLDN1, the proteolysis markers MMP3, and MMP9, and HPSE, while MMP2, VIM and ROCK-2 were downregulated. Immunocytochemistry confirmed upregulation of MMP9 and fibronectin, the latter being particular prominent after ROCK inhibition. STRING analysis confirmed an interaction of the investigated gene products at the protein level. Our results suggest that diminished Sdc-1 expression plays a role in DCIS progression to IBC through deregulation of proteolytic factors and a partial EMT.

Resveratrol impairs cellular mechanisms associated with the pathogenesis of endometriosis.

RESEARCH QUESTION: Does resveratrol exert a potent inhibitory effect on the development of endometriosis by interfering with some pivotal processes? DESIGN: In-vitro cultures of primary endometriotic stromal cells, immortalized endometrial stromal (St-T1b) and endometriotic epithelial (12Z) cells were used to assess the effects of resveratrol on endometrial cell mechanisms. The effects of resveratrol on 12Z and St-T1b cell viability were assessed by MTT assay, apoptosis by FITC Annexin V assay and cleaved caspase-3 levels and cell migration by wound healing assay. The effect of resveratrol on the expression of genes related to cell migration, angiogenesis and cell stemness was evaluated by qRT-PCR. RESULTS: Resveratrol significantly decreased cell viability (P= 0.0065 to P = 0.0180), cell migration (P < 0.001 to P = 0.0225) and increased the number of apoptotic cells (P = 0.0031 to P = 0.0432) in both cell lines. In cell lines and primary culture, the treatment reduced MMP-2/TIMP-1 (P < 0.001 to P = 0.0180), VEGF (P = 0.0052 to P = 0.0243) and Ang-1 mRNA (P < 0.001 to P = 0.0382) expression. Among the stem cell phenotype markers, resveratrol 100 µM increased mRNA expression levels of Notch-1 (P < 0.001 to P = 0.0018), KLF-4 (P = 0.0011 to P = 0.0137), SOX-2 (P < 0.001 to P = 0.0070) and TERT (P < 0.001 to P = 0.0193) in both cell lines and primary cultures. The mRNA expression level of Snail-1 increased in the cell lines (P < 0.001 to P = 0.0087), whereas OCT-4 mRNA expression increased in St-T1b (P = 0.0396) and primary cultures (P = 0.0148). Vimentin mRNA expression showed a significant upregulation in primary cultures (P < 0.001). The expression of Msi-1 (P = 0.0145) and NANOG (P = 0.0080) decreased only in St-T1b cells. CONCLUSION: Resveratrol showed inhibitory effects on cell behaviour related to the development of endometriosis by differentially affecting growth, apoptosis, migration and stem cell phenotype of endometrial and endometriotic cells in vitro.

Impact of Musashi-1 and Musashi-2 Double Knockdown on Notch Signaling and the Pathogenesis of Endometriosis.

The stem cell marker and RNA-binding protein Musashi-1 is overexpressed in endometriosis. Musashi-1-siRNA knockdown in Ishikawa cells altered the expression of stem cell related genes, such as OCT-4. To investigate the role of both human Musashi homologues (MSI-1 and MSI-2) in the pathogenesis of endometriosis, immortalized endometriotic 12-Z cells and primary endometriotic stroma cells were treated with Musashi-1- and Musashi-2-siRNA. Subsequently, the impact on cell proliferation, cell apoptosis, cell necrosis, spheroid formation, stem cell phenotype and the Notch signaling pathway was studied in vitro. Using the ENDOMET Turku Endometriosis database, the gene expression of stem cell markers and Notch signaling pathway constituents were analyzed according to localization of the endometriosis lesions. The database analysis demonstrated that expression of Musashi and Notch pathway-related genes are dysregulated in patients with endometriosis. Musashi-1/2-double-knockdown increased apoptosis and necrosis and reduced stem cell gene expression, cell proliferation, and the formation of spheroids. Musashi silencing increased the expression of the anti-proliferation mediator p21. Our findings suggest the therapeutic potential of targeting the Musashi-Notch axis. We conclude that the Musashi genes have an impact on Notch signaling and the pathogenesis of endometriosis through the downregulation of proliferation, stemness characteristics and the upregulation of apoptosis, necrosis and of the cell cycle regulator p21.

ESHRE guideline: endometriosis.

STUDY QUESTION: How should endometriosis be diagnosed and managed based on the best available evidence from published literature? SUMMARY ANSWER: The current guideline provides 109 recommendations on diagnosis, treatments for pain and infertility, management of disease recurrence, asymptomatic or extrapelvic disease, endometriosis in adolescents and postmenopausal women, prevention and the association with cancer. WHAT IS KNOWN ALREADY: Endometriosis is a chronic condition with a plethora of presentations in terms of not only the occurrence of lesions, but also the presence of signs and symptoms. The most important symptoms include pain and infertility. STUDY DESIGN SIZE DURATION: The guideline was developed according to the structured methodology for development of ESHRE guidelines. After formulation of key questions by a group of experts, literature searches and assessments were performed. Papers published up to 1 December 2020 and written in English were included in the literature review. PARTICIPANTS/MATERIALS SETTING METHODS: Based on the collected evidence, recommendations were formulated and discussed within specialist subgroups and then presented to the core guideline development group (GDG) until consensus was reached. A stakeholder review was organized after finalization of the draft. The final version was approved by the GDG and the ESHRE Executive Committee. MAIN RESULTS AND THE ROLE OF CHANCE: This guideline aims to help clinicians to apply best care for women with endometriosis. Although studies mostly focus on women of reproductive age, the guideline also addresses endometriosis in adolescents and postmenopausal women. The guideline outlines the diagnostic process for endometriosis, which challenges laparoscopy and histology as gold standard diagnostic tests. The options for treatment of endometriosis-associated pain symptoms include analgesics, medical treatments and surgery. Non-pharmacological treatments are also discussed. For management of endometriosis-associated infertility, surgical treatment and/or medically assisted reproduction are feasible. While most of the more recent studies confirm previous ESHRE recommendations, there are five topics in which significant changes to recommendations were required and changes in clinical practice are to be expected. LIMITATIONS REASONS FOR CAUTION: The guideline describes different management options but, based on existing evidence, no firm recommendations could be formulated on the most appropriate treatments. Also, for specific clinical issues, such as asymptomatic endometriosis or extrapelvic endometriosis, the evidence is too scarce to make evidence-based recommendations. WIDER IMPLICATIONS OF THE FINDINGS: The guideline provides clinicians with clear advice on best practice in endometriosis care, based on the best evidence currently available. In addition, a list of research recommendations is provided to stimulate further studies in endometriosis. STUDY FUNDING/COMPETING INTERESTS: The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings, with the literature searches and with the dissemination of the guideline. The guideline group members did not receive payments. C.M.B. reports grants from Bayer Healthcare and the European Commission; Participation on a Data Safety Monitoring Board or Advisory Board with ObsEva (Data Safety Monitoring Group) and Myovant (Scientific Advisory Group). A.B. reports grants from FEMaLE executive board member and European Commission Horizon 2020 grant; consulting fees from Ethicon Endo Surgery, Medtronic; honoraria for lectures from Ethicon; and support for meeting attendance from Gedeon Richter; A.H. reports grants from MRC, NIHR, CSO, Roche Diagnostics, Astra Zeneca, Ferring; Consulting fees from Roche Diagnostics, Nordic Pharma, Chugai and Benevolent Al Bio Limited all paid to the institution; a pending patent on Serum endometriosis biomarker; he is also Chair of TSC for STOP-OHSS and CERM trials. O.H. reports consulting fees and speaker's fees from Gedeon Richter and Bayer AG; support for attending meetings from Gedeon-Richter, and leadership roles at the Finnish Society for Obstetrics and Gynecology and the Nordic federation of the societies of obstetrics and gynecology. L.K. reports consulting fees from Gedeon Richter, AstraZeneca, Novartis, Dr KADE/Besins, Palleos Healthcare, Roche, Mithra; honoraria for lectures from Gedeon Richter, AstraZeneca, Novartis, Dr KADE/Besins, Palleos Healthcare, Roche, Mithra; support for attending meetings from Gedeon Richter, AstraZeneca, Novartis, Dr KADE/Besins, Palleos Healthcare, Roche, Mithra; he also has a leadership role in the German Society of Gynecological Endocrinology (DGGEF). M.K. reports grants from French Foundation for Medical Research (FRM), Australian Ministry of Health, Medical Research Future Fund and French National Cancer Institute; support for meeting attendance from European Society for Gynaecological Endoscopy (ESGE), European Congress on Endometriosis (EEC) and ESHRE; She is an advisory Board Member, FEMaLe Project (Finding Endometriosis Using Machine Learning), Scientific Committee Chair for the French Foundation for Research on Endometriosis and Scientific Committee Chair for the ComPaRe-Endometriosis cohort. A.N. reports grants from Merck SA and Ferring; speaker fees from Merck SA and Ferring; support for meeting attendance from Merck SA; Participation on a Data Safety Monitoring Board or Advisory Board with Nordic Pharma and Merck SA; she also is a board member of medical advisory board, Endometriosis Society, the Netherlands (patients advocacy group) and an executive board member of the World Endometriosis Society. E.S. reports grants from National Institute for Health Research UK, Rosetrees Trust, Barts and the London Charity; Royalties from De Gruyter (book editor); consulting fees from Hologic; speakers fees from Hologic, Johnson & Johnson, Medtronic, Intuitive, Olympus and Karl Storz; Participation in the Medicines for Women's Health Expert Advisory Group with Medicines and Healthcare Products Regulatory Agency (MHRA); he is also Ambassador for the World Endometriosis Society. C.T. reports grants from Merck SA; Consulting fees from Gedeon Richter, Nordic Pharma and Merck SA; speaker fees from Merck SA, all paid to the institution; and support for meeting attendance from Ferring, Gedeon Richter and Merck SA. The other authors have no conflicts of interest to declare. DISCLAIMER: This guideline represents the views of ESHRE, which were achieved after careful consideration of the scientific evidence available at the time of preparation. In the absence of scientific evidence on certain aspects, a consensus between the relevant ESHRE stakeholders has been obtained. Adherence to these clinical practice guidelines does not guarantee a successful or specific outcome, nor does it establish a standard of care. Clinical practice guidelines do not replace the need for application of clinical judgement to each individual presentation, nor variations based on locality and facility type. ESHRE makes no warranty, express or implied, regarding the clinical practice guidelines and specifically excludes any warranties of merchantability and fitness for a particular use or purpose (Full disclaimer available at www.eshre.eu/guidelines.).

Differential Impact of Membrane-Bound and Soluble Forms of the Prognostic Marker Syndecan-1 on the Invasiveness, Migration, Apoptosis, and Proliferation of Cervical Cancer Cells.

Cervical cancer ranks fourth among the most commonly diagnosed malignant tumors in women worldwide. Previously published evidence suggested a possible connection between the expression of the membrane-bound heparan sulfate proteoglycan syndecan-1 (Sdc-1) and the development of cervical carcinoma. Sdc-1 serves as a matrix receptor and coreceptor for receptor tyrosine kinases and additional signaling pathways. It influences cell proliferation, adhesion, and migration and is seen as a modulator of the tumor microenvironment. Following proteolytic cleavage of its extracellular domain in a process called shedding, Sdc-1 can act as a paracrine effector. The loss of Sdc-1 expression is associated with low differentiation of cervical carcinoma and with an increased rate of lymph node metastases. Here, we analyzed the clinical impact of Sdc-1 expression by analysis of public gene expression datasets and studied the effect of an overexpression of Sdc-1 and its membrane-bound and soluble forms on the malignant properties of the human cervical carcinoma cell line HeLa through functional analysis. For this purpose, the HeLa cells were stably transfected with the control plasmid pcDNA3.1 and three different Sdc-1-DNA constructs,encoding wild-type, permanently membrane-bound, and constitutively soluble Sdc-1. In clinical specimens, Sdc-1 mRNA was more highly expressed in local tumor tissues than in normal and metastatic cervical cancer tissues. Moreover, high Sdc-1 expression correlated with a poor prognosis in Kaplan-Meier survival analysis, suggesting the important role of Sdc-1 in the progression of this type of cancer. In vitro, we found that the soluble, as well as the permanently membrane-bound forms of Sdc-1 modulated the proliferation and the cell cycle, while membrane-bound Sdc1 regulated HeLa cell apoptosis. The overexpression of Sdc-1 and its soluble form increased invasiveness. In vitro scratch/wound healing assay, showed reduced Sdc-1-dependent cell motility which was linked to the Rho-GTPase signaling pathway. In conclusion, in cervical cancer Sdc-1 modulates pathogenetically relevant processes, which depend on the membrane-association of Sdc-1.

Diagnosis, Therapy and Follow-up of Cervical Cancer. Guideline of the DGGG, DKG and DKH (S3-Level, AWMF Registry No. 032/033OL, May 2021) - Part 1 with Recommendations on Epidemiology, Screening, Diagnostics and Therapy.

Aim This update of the interdisciplinary S3 guideline on the Diagnosis, Therapy and Follow-up of Cervical Cancer (AWMF Registry No. 032/033OL) was published in March 2021. This updated guideline was funded by German Cancer Aid (Deutsche Krebshilfe) as part of the German Guideline Program in Oncology. The guideline was coordinated by the German Society of Gynecology and Obstetrics ( Deutsche Gesellschaft für Gynäkologie und Geburtshilfe , DGGG) and the Working Group on Gynecological Oncology ( Arbeitsgemeinschaft Gynäkologische Onkologie , AGO) of the German Cancer Society ( Deutsche Krebsgesellschaft , DKG). Method The process of updating the S3 guideline dating from 2014 was based on an appraisal of the available evidence using the criteria of evidence-based medicine, adaptations of existing evidence-based national and international guidelines or - if evidence was lacking - on a consensus of the specialists involved in compiling the update. After an initial review of the current literature was carried out according to a prescribed algorithm, several areas were identified which, in contrast to the predecessor version from September 2014, required new recommendations or statements which took account of more recently published literature and the appraisal of the new evidence. Recommendations The short version of this guideline consists of recommendations and statements on the epidemiology, screening, diagnostic workup and therapy of patients with cervical cancer. The most important new aspects included in this updated guideline include the newly published FIGO classification of 2018, the radical open surgery approach for cervical cancers up to FIGO stage IB1, and use of the sentinel lymph node technique for tumors ≤ 2 cm. Other changes include the use of PET-CT, new options in radiotherapy (e.g., intensity-modulated radiotherapy, image-guided adaptive brachytherapy), and drug therapies to treat recurrence or metastasis.

Diagnosis, Therapy and Follow-up of Cervical Cancer. Guideline of the DGGG, DKG and DKH (S3-Level, AWMF Registry No. 032/033OL, May 2021) - Part 2 with Recommendations on Psycho-oncology, Rehabilitation, Follow-up, Recurrence, Palliative Therapy and Healthcare Facilities.

Aim This is an update of the interdisciplinary S3-guideline on the Diagnosis, Therapy and Follow-up of Cervical Cancer (AWMF Registry No. 032/033OL), published in March 2021. The work on the updated guideline was funded by German Cancer Aid (Deutsche Krebshilfe) as part of the German Guideline Program in Oncology. The guideline was coordinated by the German Society of Gynecology and Obstetrics ( Deutsche Gesellschaft für Gynäkologie und Geburtshilfe , DGGG) and the Working Group on Gynecological Oncology ( Arbeitsgemeinschaft Gynäkologische Onkologie , AGO) of the German Cancer Society ( Deutsche Krebsgesellschaft , DKG). Method The process used to update the 2014 S3-guideline was based on an appraisal of the available evidence using the criteria of evidence-based medicine, adaptations of existing evidence-based national and international guidelines or - if evidence was lacking - on the consensus of the specialists involved in compiling the update. After an initial review of the current literature was carried out according to a prescribed algorithm, several areas were identified which, in contrast to the predecessor version from September 2014, required new recommendations or statements which would take account of more recently published literature and the recent appraisal of new evidence. Recommendations The short version of this guideline consists of recommendations and statements on palliative therapy and follow-up of patients with cervical cancer. The most important aspects included in this updated guideline are the new FIGO classification published in 2018, the radical open surgery approach used to treat cervical cancer up to FIGO stage IB1, and the use of the sentinel lymph node technique for tumors ≤ 2 cm. Other changes include the use of PET-CT, new options in radiotherapy (e.g., intensity-modulated radiotherapy, image-guided adaptive brachytherapy), and drug therapies to treat recurrence or metastasis.