L. Kiesel

What is the optimal management of women with endometriosis based on the best available evidence in the literature? Using the structured methodology of the Manual for ESHRE Guideline Development, 83 recommendations were formulated that answered the 22 key questions on optimal management of women with endometriosis. The European Society of Human Reproduction and Embryology (ESHRE) guideline for the diagnosis and treatment of endometriosis (2005) has been a reference point for best clinical care in endometriosis for years, but this guideline was in need of updating. This guideline was produced by a group of experts in the field using the methodology of the Manual for ESHRE Guideline Development, including a thorough systematic search of the literature, quality assessment of the included papers up to January 2012 and consensus within the guideline group on all recommendations. To ensure input from women with endometriosis, a patient representative was part of the guideline development group. In addition, patient and additional clinical input was collected during the scoping and review phase of the guideline. NA. The guideline provides 83 recommendations on diagnosis of endometriosis and on the treatment of endometriosis-associated pain and infertility, on the management of women in whom the disease is found incidentally (without pain or infertility), on prevention of recurrence of disease and/or painful symptoms, on treatment of menopausal symptoms in patients with a history of endometriosis and on the possible association of endometriosis and malignancy. We identified several areas in care of women with endometriosis for which robust evidence is lacking. These areas were addressed by formulating good practice points (GPP), based on the expert opinion of the guideline group members. Since 32 out of the 83 recommendations for the management of women with endometriosis could not be based on high level evidence and therefore were GPP, the guideline group formulated research recommendations to guide future research with the aim of increasing the body of evidence. The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings, with the literature searches and with the implementation of the guideline. The guideline group members did not receive payment. All guideline group members disclosed any relevant conflicts of interest (see Conflicts of interest). NA.

What is the global consensus on the classification of endometriosis that considers the views of women with endometriosis?We have produced an international consensus statement on the classification of endometriosis through systematic appraisal of evidence and a consensus process that included representatives of national and international, medical and non-medical societies, patient organizations, and companies with an interest in endometriosis.Classification systems of endometriosis, developed by several professional organizations, traditionally have been based on lesion appearance, pelvic adhesions, and anatomic location of disease. One system predicts fertility outcome and none predicts pelvic pain, response to medications, disease recurrence, risks for associated disorders, quality of life measures, and other endpoints important to women and health care providers for guiding appropriate therapeutic options and prognosis.A consensus meeting, in conjunction with pre- and post-meeting processes, was undertaken.A consensus meeting was held on 30 April 2014 in conjunction with the World Endometriosis Society's 12th World Congress on Endometriosis. Rigorous pre- and post-meeting processes, involving 55 representatives of 29 national and international, medical and non-medical organizations from a range of disciplines, led to this consensus statement.A total of 28 consensus statements were made. Of all, 10 statements had unanimous consensus, however none of the statements was made without expression of a caveat about the strength of the statement or the statement itself. Two statements did not achieve majority consensus. The statements covered women's priorities, aspects of classification, impact of low resources, as well as all the major classification systems for endometriosis. Until better classification systems are developed, we propose a classification toolbox (that includes the revised American Society for Reproductive Medicine and, where appropriate, the Enzian and Endometriosis Fertility Index staging systems), that may be used by all surgeons in each case of surgery undertaken for women with endometriosis. We also propose wider use of the World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project surgical and clinical data collection tools for research to improve classification of endometriosis in the future, of particular relevance when surgery is not undertaken.This consensus process differed from that of formal guideline development, although based on the same available evidence. A different group of international experts from those participating in this process may have yielded subtly different consensus statements.This is the first time that a large, global, consortium-representing 29 major stake-holding organizations, from 19 countries - has convened to systematically evaluate the best available evidence on the classification of endometriosis and reach consensus. In addition to 21 international medical organizations and companies, representatives from eight national endometriosis organizations were involved, including lay support groups, thus generating and including input from women who suffer from endometriosis in an endeavour to keep uppermost the goal of optimizing quality of life for women with endometriosis.The World Endometriosis Society convened and hosted the consensus meeting. Financial support for participants to attend the meeting was provided by the organizations that they represented. There was no other specific funding for this consensus process. Mauricio Abrao is an advisor to Bayer Pharma, and a consultant to AbbVie and AstraZeneca; G David Adamson is the Owner of Advanced Reproductive Care Inc and Ziva and a consultant to Bayer Pharma, Ferring, and AbbVie; Deborah Bush has received travel grants from Fisher & Paykel Healthcare and Bayer Pharmaceuticals; Linda Giudice is a consultant to AbbVie, Juniper Pharmaceutical, and NextGen Jane, holds research grant from the NIH, is site PI on a clinical trial sponsored by Bayer, and is a shareholder in Merck and Pfizer; Lone Hummelshoj is an unpaid consultant to AbbVie; Neil Johnson has received conference expenses from Bayer Pharma, Merck-Serono, and MSD, research funding from AbbVie, and is a consultant to Vifor Pharma and Guerbet; Jörg Keckstein has received a travel grant from AbbVie; Ludwig Kiesel is a consultant to Bayer Pharma, AbbVie, AstraZeneca, Gedeon Richter, and Shionogi, and holds a research grant from Bayer Pharma; Luk Rombauts is an advisor to MSD, Merck Serono, and Ferring, and a shareholder in Monash IVF. The following have declared that they have nothing to disclose: Kathy Sharpe Timms; Rulla Tamimi; Hugh Taylor.N/A.

How should endometriosis be diagnosed and managed based on the best available evidence from published literature?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.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.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.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.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.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.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.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.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.).

STUDY QUESTIONIs there a global consensus on the management of endometriosis that considers the views of women with endometriosis?

We evaluated discordance in expression measurements for estrogen receptor (ER), progesterone receptor (PR), and HER2 between primary and recurrent tumors in patients with recurrent breast cancer and its effect on prognosis.A total of 789 patients with recurrent breast cancer were studied. ER, PR, and HER2 status were determined by immunohistochemistry (IHC) and/or FISH. Repeat markers for ER, PR, and HER2 were available in 28.9%, 27.6%, and 70.0%, respectively. Primary and recurrent tumors were classified as triple receptor-negative breast cancer (TNBC) or receptor-positive breast cancer (RPBC, i.e. expressing at least one receptor). Discordance was correlated with clinical/pathological parameters.Discordance for ER, PR, and HER2 was 18.4%, 40.3%, and 13.6%, respectively. Patients with concordant RPBC had significantly better post-recurrence survival (PRS) than discordant cases; patients with discordant receptor status had similarly unfavorable survival as patients with concordant TNBC. IHC scores for ER and PR showed weak concordance between primary and recurrent tumors. Concordance of HER2-FISH scores was higher.Concordance of quantitative hormone receptor measurements between primary and recurrent tumors is modest consistent with suboptimal reproducibility of measurement methods, particularly for IHC. Discordant cases have poor survival probably due to inappropriate use of targeted therapies. However, biological change in clinical phenotype cannot be completely excluded.

State-of-the art therapy for recurrent ovarian cancer suitable for platinum-based re-treatment includes bevacizumab-containing combinations (eg, bevacizumab combined with carboplatin-paclitaxel or carboplatin-gemcitabine) or the most active non-bevacizumab regimen: carboplatin-pegylated liposomal doxorubicin. The aim of this head-to-head trial was to compare a standard bevacizumab-containing regimen versus carboplatin-pegylated liposomal doxorubicin combined with bevacizumab.This multicentre, open-label, randomised, phase 3 trial, was done in 159 academic centres in Germany, France, Australia, Austria, and the UK. Eligible patients (aged ≥18 years) had histologically confirmed epithelial ovarian, primary peritoneal, or fallopian tube carcinoma with first disease recurrence more than 6 months after first-line platinum-based chemotherapy, and an Eastern Cooperative Oncology Group performance status of 0-2. Patients were stratified by platinum-free interval, residual tumour, previous antiangiogenic therapy, and study group language, and were centrally randomly assigned 1:1 using randomly permuted blocks of size two, four, or six to receive six intravenous cycles of bevacizumab (15 mg/kg, day 1) plus carboplatin (area under the concentration curve [AUC] 4, day 1) plus gemcitabine (1000 mg/m2, days 1 and 8) every 3 weeks or six cycles of bevacizumab (10 mg/kg, days 1 and 15) plus carboplatin (AUC 5, day 1) plus pegylated liposomal doxorubicin (30 mg/m2, day 1) every 4 weeks, both followed by maintenance bevacizumab (15 mg/kg every 3 weeks in both groups) until disease progression or unacceptable toxicity. There was no masking in this open-label trial. The primary endpoint was investigator-assessed progression-free survival according to Response Evaluation Criteria in Solid Tumors version 1.1. Efficacy data were analysed in the intention-to-treat population. Safety was analysed in all patients who received at least one dose of study drug. This completed study is registered with ClinicalTrials.gov, NCT01837251.Between Aug 1, 2013, and July 31, 2015, 682 eligible patients were enrolled, of whom 345 were randomly assigned to receive carboplatin-pegylated liposomal doxorubicin-bevacizumab (experimental group) and 337 were randomly assigned to receive carboplatin-gemcitabine-bevacizumab (standard group). Median follow-up for progression-free survival at data cutoff (July 10, 2018) was 12·4 months (IQR 8·3-21·7) in the experimental group and 11·3 months (8·0-18·4) in the standard group. Median progression-free survival was 13·3 months (95% CI 11·7-14·2) in the experimental group versus 11·6 months (11·0-12·7) in the standard group (hazard ratio 0·81, 95% CI 0·68-0·96; p=0·012). The most common grade 3 or 4 adverse events were hypertension (88 [27%] of 332 patients in the experimental group vs 67 [20%] of 329 patients in the standard group) and neutropenia (40 [12%] vs 73 [22%]). Serious adverse events occurred in 33 (10%) of 332 patients in the experimental group and 28 (9%) of 329 in the standard group. Treatment-related deaths occurred in one patient in the experimental group (<1%; large intestine perforation) and two patients in the standard group (1%; one case each of osmotic demyelination syndrome and intracranial haemorrhage).Carboplatin-pegylated liposomal doxorubicin-bevacizumab is a new standard treatment option for platinum-eligible recurrent ovarian cancer.F Hoffmann-La Roche.

Early metastasis in node-negative breast cancer indicates that breast cancer cells obviously can bypass the lymph nodes and disseminate directly hematogenous to distant organs. For this purpose, we evaluated the prognostic value of blood-borne, HER2-positive circulating tumor cells (CTC) in the peripheral blood from 42 breast cancer patients with a median follow-up of 95 months.Cells were isolated by the patented combined buoyant density gradient and immunomagnetic separation procedure and analyzed by immunocytochemistry.We detected one to eight CTCs in the peripheral blood of 17 of 35 patients (48.6%) presenting no overt metastasis. As a positive control, 7 of 7 (100%) patients with metastatic disease presented positive. Healthy persons and patients (n = 32) operated for nonmalignant diseases presented negative for CTCs. The presence and frequency of HER2-positive CTCs correlated with a significantly decreased disease-free survival (P < 0.005) and overall survival (P < 0.05). Interestingly, in 12 patients with HER2-positive CTCs, the primary tumor was negative for HER2 as assessed by immunohistochemical score and fluorescence in situ hybridization.This study provides some evidence of a prognostic effect of HER2-positive CTCs in stage I to III breast cancer. Future studies have to determine the outcome of patients treated with HER2-targeting therapies with respect to HER2-positive CTC levels because it is not unlikely that high levels of HER2-positive CTCs reflect the activity of the tumor and may predict response to trastuzumab.

Micro RNAs are small non-coding RNAs, which regulate fundamental cellular and developmental processes at the transcriptional and translational level. In breast cancer, miR-145 expression is downregulated compared with healthy control tissue. As several predicted targets of miR-145 potentially regulate cell motility, we aimed at investigating a potential role for miR-145 in breast cancer cell motility and invasiveness. Assisted by Affymetrix array technology, we demonstrate that overexpression of miR-145 in MDA-MB-231, MCF-7, MDA-MB-468 and SK-BR-3 breast cancer cells and in Ishikawa endometrial carcinoma cells leads to a downregulation of the cell–cell adhesion protein JAM-A and of the actin bundling protein fascin. Moreover, podocalyxin and Serpin E1 mRNA levels were downregulated, and gamma-actin, transgelin and MYL9 were upregulated upon miR-145 overexpression. These miR-145-dependent expression changes drastically decreased cancer cell motility, as revealed by time-lapse video microscopy, scratch wound closure assays and matrigel invasion assays. Immunofluorescence microscopy demonstrated restructuring of the actin cytoskeleton and a change in cell morphology by miR-145 overexpression, resulting in a more cortical actin distribution, and reduced actin stress fiber and filopodia formation. Nuclear rotation was observed in 10% of the pre-miR-145 transfected MDA-MB-231 cells, accompanied by a reduction of perinuclear actin. Luciferase activation assays confirmed direct miR-145-dependent regulation of the 3′UTR of JAM-A, whereas siRNA-mediated knockdown of JAM-A expression resulted in decreased motility and invasiveness of MDA-MB-231 and MCF-7 breast cancer cells. Our data identify JAM-A and fascin as novel targets of miR-145, firmly establishing a role for miR-145 in modulating breast cancer cell motility. Our data provide a rationale for future miR-145-targeted approaches of antimetastatic cancer therapy.

Abstract Adult stem cells are thought to be responsible for the high regenerative capacity of the human endometrium, and have been implicated in the pathology of endometriosis and endometrial carcinoma. The RNA‐binding protein Musashi‐1 is associated with maintenance and asymmetric cell division of neural and epithelial progenitor cells. We investigated expression and localization of Musashi‐1 in endometrial, endometriotic and endometrial carcinoma tissue specimens of 46 patients. qPCR revealed significantly increased Musashi‐1 mRNA expression in the endometrium compared to the myometrium. Musashi‐1 protein expression presented as nuclear or cytoplasmic immunohistochemical staining of single cells in endometrial glands, and of single cells and cell groups in the endometrial stroma. Immunofluorescence microscopy revealed colocalization of Musashi‐1 with its molecular target Notch‐1 and telomerase. In proliferative endometrium, the proportion of Musashi‐1‐positive cells in the basalis layer was significantly increased 1.5‐fold in the stroma, and three‐fold in endometrial glands compared to the functionalis. The number of Musashi‐1 expressing cell groups was significantly increased (four‐fold) in proliferative compared to secretory endometrium. Musashi‐1 expressing stromal cell and cell group numbers were significantly increased (five‐fold) in both endometriotic and endometrial carcinoma tissue compared to secretory endometrium. A weak to moderate, diffuse cytoplasmic glandular staining was observed in 50% of the endometriosis cases and in 75% of the endometrioid carcinomas compared to complete absence in normal endometrial samples. Our results emphasize the role of Musashi‐1‐expressing endometrial progenitor cells in proliferating endometrium, endometriosis and endometrioid uterine carcinoma, and support the concept of a stem cell origin of endometriosis and endometrial carcinoma. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

The p.N680S sequence variation of the follicle-stimulating hormone (FSH) receptor gene was previously shown to influence the ovarian response to FSH in normo-ovulatory women undergoing controlled ovarian hyperstimulation. In this prospective, randomized, controlled study, we tested whether the same daily dose of FSH results in lower levels of oestradiol in women homozygous for the p.N680S sequence variation, and whether the difference can be overcome by higher FSH doses. Women undergoing controlled ovarian hyperstimulation for in vitro fertilization or intracytoplasmic sperm injection and homozygous for the wild-type or for the p.N680S FSH receptor were randomly assigned to group I (Ser/Ser, n=24), receiving an FSH dose of 150 U/day, or group II (Ser/Ser, n=25), receiving an FSH dose of 225 U/day. In group III (Asn/Asn, n=44), the FSH dose was 150 U/day. Age and basal FSH levels were not different between groups. At ovulation induction, total FSH doses were comparable in group I (1631±96 U) and group III (1640±57 U) but significantly higher in group II (2421±112 U) (P<0.001). Peak oestradiol levels on the day of human chorionic gonadotrophin (hCG) administration were significantly lower in group I (5680±675 pmol/l) compared to group III (8679±804 pmol/l) (P=0.028). Increasing the FSH dose from 150 to 225 U/day overcame the lower oestradiol response in women with Ser/Ser (group II, 7804±983 pmol/l). In women undergoing controlled ovarian hyperstimulation, the p.N680S sequence variation results in lower oestradiol levels following FSH stimulation. This lower FSH receptor sensitivity can be overcome by higher FSH doses.

The heparan sulfate proteoglycan syndecan-1 (Sdc1) modulates cell proliferation, adhesion, migration and angiogenesis. Proteinase-mediated shedding converts Sdc1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. In breast carcinomas, Sdc1 overexpression correlates with poor prognosis and an aggressive phenotype. To distinguish between the roles of membrane-bound and shed forms of Sdc1 in breast cancer progression, human MCF-7 breast cancer cells were stably transfected with plasmids overexpressing wild-type (WT), constitutively shed and uncleavable forms of Sdc1. Overexpression of WT Sdc1 increased cell proliferation, whereas overexpression of constitutively shed Sdc1 decreased proliferation. Fibroblast growth factor-2-mediated mitogen-activated protein kinase signaling was reduced following small-interfering RNA (siRNA)-mediated knockdown of Sdc1 expression. Constitutively, membrane-bound Sdc1 inhibited invasiveness, whereas soluble Sdc1 promoted invasion of MCF-7 cells into matrigel matrices. The latter effect was reversed by the matrix metalloproteinase inhibitors N -isobutyl- N -(4-methoxyphenylsufonyl) glycyl hydroxamic acid and tissue inhibitor of metalloproteinase (TIMP)-1. Affymetrix microarray analysis identified TIMP-1, Furin and urokinase-type plasminogen activator receptor as genes differentially regulated in soluble Sdc1-overexpressing cells. Endogenous TIMP-1 expression was reduced in cells overexpressing soluble Sdc1 and increased in those overexpressing the constitutively membrane-bound Sdc1. Moreover, E-cadherin protein expression was downregulated in cells overexpressing soluble Sdc1. Our results suggest that the soluble and membrane-bound forms of Sdc1 play different roles at different stages of breast cancer progression. Proteolytic conversion of Sdc1 from a membrane-bound into a soluble molecule marks a switch from a proliferative to an invasive phenotype, with implications for breast cancer diagnostics and potential glycosaminoglycan-based therapies.

To standardize the recording of surgical phenotypic information on endometriosis and related sample collections obtained at laparoscopy, allowing large-scale collaborative research into the condition.An international collaboration involving 34 clinical/academic centers and three industry collaborators from 16 countries.Two workshops were conducted in 2013, bringing together 54 clinical, academic, and industry leaders in endometriosis research and management worldwide.None.A postsurgical scoring sheet containing general and gynecological patient and procedural information, extent of disease, the location and type of endometriotic lesion, and any other findings was developed during several rounds of review. Comments and any systematic surgical data collection tools used in the reviewers' centers were incorporated.The development of a standard recommended (SSF) and minimum required (MSF) form to collect data on the surgical phenotype of endometriosis.SSF and MSF include detailed descriptions of lesions, modes of procedures and sample collection, comorbidities, and potential residual disease at the end of surgery, along with previously published instruments such as the revised American Society for Reproductive Medicine and Endometriosis Fertility Index classification tools for comparison and validation.This is the first multicenter, international collaboration between academic centers and industry addressing standardization of phenotypic data collection for a specific disease. The Endometriosis Phenome and Biobanking Harmonisation Project SSF and MSF are essential tools to increase our understanding of the pathogenesis of endometriosis by allowing large-scale collaborative research into the condition.

ObjectiveTo harmonize standard operating procedures (SOPs) and standardize the recording of associated data for collection, processing, and storage of fluid biospecimens relevant to endometriosis.DesignAn international collaboration involving 34 clinical/academic centers and 3 industry collaborators from 16 countries on 5 continents.SettingIn 2013, 2 workshops were conducted, followed by global consultation, bringing together 54 leaders in endometriosis research and sample processing worldwide.Patient(s)None.Intervention(s)Consensus SOPs were based on: [1] systematic comparison of SOPs from 18 global centers collecting fluid samples from women with and without endometriosis on a medium/large scale (publication on >100 cases), [2] literature evidence where available, or consultation with laboratory experts otherwise, and [3] several global consultation rounds.Main Outcome Measure(s)Standard recommended and minimum required SOPs for biofluid collection, processing, and storage in endometriosis research.Result(s)We developed recommended standard and minimum required SOPs for the collection, processing, and storage of plasma, serum, saliva, urine, endometrial/peritoneal fluid, and menstrual effluent, and a biospecimen data-collection form necessary for interpretation of sample-derived results.Conclusion(s)The Endometriosis Phenome and Biobanking Harmonisation Project SOPs allow endometriosis research centers to decrease variability in biofluid sample results, facilitating between-center comparisons and collaborations. The procedures are also relevant to research into other female conditions involving biofluid samples subject to cyclic reproductive influences. The consensus SOPs are based on the best available evidence; areas with limited evidence are identified as requiring further pilot studies. The SOPs will be reviewed based on investigator feedback, and through systematic tri-annual follow-up. Updated versions will be made available at: endometriosisfoundation.org/ephect. To harmonize standard operating procedures (SOPs) and standardize the recording of associated data for collection, processing, and storage of fluid biospecimens relevant to endometriosis. An international collaboration involving 34 clinical/academic centers and 3 industry collaborators from 16 countries on 5 continents. In 2013, 2 workshops were conducted, followed by global consultation, bringing together 54 leaders in endometriosis research and sample processing worldwide. None. Consensus SOPs were based on: [1] systematic comparison of SOPs from 18 global centers collecting fluid samples from women with and without endometriosis on a medium/large scale (publication on >100 cases), [2] literature evidence where available, or consultation with laboratory experts otherwise, and [3] several global consultation rounds. Standard recommended and minimum required SOPs for biofluid collection, processing, and storage in endometriosis research. We developed recommended standard and minimum required SOPs for the collection, processing, and storage of plasma, serum, saliva, urine, endometrial/peritoneal fluid, and menstrual effluent, and a biospecimen data-collection form necessary for interpretation of sample-derived results. The Endometriosis Phenome and Biobanking Harmonisation Project SOPs allow endometriosis research centers to decrease variability in biofluid sample results, facilitating between-center comparisons and collaborations. The procedures are also relevant to research into other female conditions involving biofluid samples subject to cyclic reproductive influences. The consensus SOPs are based on the best available evidence; areas with limited evidence are identified as requiring further pilot studies. The SOPs will be reviewed based on investigator feedback, and through systematic tri-annual follow-up. Updated versions will be made available at: endometriosisfoundation.org/ephect.

FSH is essential for follicular maturation. Data from ovarian hyperstimulation cycles suggest that FSH action is attenuated by a frequent single nucleotide polymorphism of the FSH receptor gene exchanging Asn for Ser at codon 680.We hypothesized that the FSH receptor genotype influences menstrual cycle dynamics.Menstrual cycle was monitored from the midluteal phase through ovulation until the consecutive menstruation.The study was conducted at the University research center.Women homozygous for the Asn680 (n = 12) and Ser680 (n = 9) variants with normal menstrual cycles volunteered for the study.There were no interventions.Follicular growth, serum LH, FSH, estradiol, progesterone, inhibin A, inhibin B and antimullerian hormone were measured.During the luteo-follicular transition, serum levels of estradiol, progesterone, and inhibin A were significantly lower, and FSH started to rise earlier in the Ser680/Ser680 group. FSH levels were steadily and significantly higher, and the mean area under the FSH curve was 31% greater in this group (P < 0.002). No differences were observed in estradiol, inhibin B, and growth velocities of dominant follicles. The time from luteolysis to ovulation was significantly longer in women with the Ser680/Ser680 (13.6 +/- 1.01 d) compared with Asn680/Asn680 (11.3 +/- 0.61 d, P < 0.05) genotype with a significant difference in total menstrual cycle length (29.3 vs. 27.0 d, respectively; P < 0.05).The FSH receptor Ser680/Ser680 genotype is associated with higher ovarian threshold to FSH, decreased negative feedback of luteal secretion to the pituitary during the intercycle transition, and longer menstrual cycles.

Abstract microRNAs are small endogenous noncoding RNAs, which post‐transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan‐1, a predicted target of the oncomiR miR‐10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR‐10b and syndecan‐1, MDA‐MB‐231 and MCF‐7 breast cancer cells were transiently transfected with pre‐miR‐10b, syndecan‐1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time‐lapse video microscopy. miR‐10b overexpression induced post‐transcriptional downregulation of syndecan‐1, as demonstrated by quantitative real‐time PCR (qPCR), flow cytometry, and 3′UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan‐1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan‐1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan‐1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan‐1‐deficient cells revealed upregulation of ATF‐2, COX‐2, cadherin‐11, vinculin, actin γ 2, MYL9, transgelin‐1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E‐cadherin, CLDN1, p21WAF/CIP, cyclin‐dependent kinase 6, TLR‐4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin‐2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase‐dependent motility of syndecan‐1‐depleted cells, which displayed increased filopodia formation. We conclude that syndecan‐1 is a novel target of the oncomiR miR‐10b. Rho‐GTPase‐dependent modulation of cytoskeletal function and downregulation of E‐cadherin expression are identified as relevant effectors of the miR‐10b‐syndecan‐1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.

ObjectiveTo harmonize standard operating procedures (SOPs) and standardize the recording of associated data for collection, processing, and storage of human tissues relevant to endometriosis.DesignAn international collaboration involving 34 clinical/academic centers and three industry collaborators from 16 countries on five continents.SettingIn 2013, two workshops were conducted followed by global consultation, bringing together 54 leaders in endometriosis research and sample processing from around the world.Patient(s)None.Intervention(s)Consensus SOPs were based on: 1) systematic comparison of SOPs from 24 global centers collecting tissue samples from women with and without endometriosis on a medium or large scale (publication on >100 cases); 2) literature evidence where available, or consultation with laboratory experts otherwise; and 3) several global consultation rounds.Main Outcome Measure(s)Standard recommended and minimum required SOPs for tissue collection, processing, and storage in endometriosis research.Result(s)We developed “recommended standard” and “minimum required” SOPs for the collection, processing, and storage of ectopic and eutopic endometrium, peritoneum, and myometrium, and a biospecimen data collection form necessary for interpretation of sample-derived results.Conclusion(s)The EPHect SOPs allow endometriosis research centers to decrease variability in tissue-based results, facilitating between-center comparisons and collaborations. The procedures are also relevant to research into other gynecologic conditions involving endometrium, myometrium, and peritoneum. The consensus SOPs are based on the best available evidence; areas with limited evidence are identified as requiring further pilot studies. The SOPs will be reviewed based on investigator feedback and through systematic triannual follow-up. Updated versions will be made available at: http://endometriosisfoundation.org/ephect. To harmonize standard operating procedures (SOPs) and standardize the recording of associated data for collection, processing, and storage of human tissues relevant to endometriosis. An international collaboration involving 34 clinical/academic centers and three industry collaborators from 16 countries on five continents. In 2013, two workshops were conducted followed by global consultation, bringing together 54 leaders in endometriosis research and sample processing from around the world. None. Consensus SOPs were based on: 1) systematic comparison of SOPs from 24 global centers collecting tissue samples from women with and without endometriosis on a medium or large scale (publication on >100 cases); 2) literature evidence where available, or consultation with laboratory experts otherwise; and 3) several global consultation rounds. Standard recommended and minimum required SOPs for tissue collection, processing, and storage in endometriosis research. We developed “recommended standard” and “minimum required” SOPs for the collection, processing, and storage of ectopic and eutopic endometrium, peritoneum, and myometrium, and a biospecimen data collection form necessary for interpretation of sample-derived results. The EPHect SOPs allow endometriosis research centers to decrease variability in tissue-based results, facilitating between-center comparisons and collaborations. The procedures are also relevant to research into other gynecologic conditions involving endometrium, myometrium, and peritoneum. The consensus SOPs are based on the best available evidence; areas with limited evidence are identified as requiring further pilot studies. The SOPs will be reviewed based on investigator feedback and through systematic triannual follow-up. Updated versions will be made available at: http://endometriosisfoundation.org/ephect.

Background Polycystic ovary syndrome (PCOS) is the most common cause of infrequent periods (oligomenorrhoea) and absence of periods (amenorrhoea). It affects about 4% to 8% of women worldwide and often leads to anovulatory subfertility. 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). Objectives To evaluate the effectiveness and safety of aromatase inhibitors for subfertile women with anovulatory PCOS for ovulation induction followed by timed intercourse or intrauterine insemination (IUI). Search methods We searched the following sources from inception to November 2017 to identify relevant randomised controlled trials (RCTs): the Cochrane Gynaecology and Fertility Group Specialised Register, the Cochrane Central Register of Controlled Trials, MEDLINE, Embase, PsycINFO, Pubmed, LILACS, Web of Knowledge, the World Health Organization (WHO) clinical trials register and Clinicaltrials.gov. We also searched the references of relevant articles. 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. We pooled studies 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 and OHSS. Secondary outcomes were clinical pregnancy, miscarriage and multiple pregnancy. We assessed the quality of the evidence for each comparison using GRADE methods. Main results This is a substantive update of a previous review. We identified 16 additional studies for the 2018 update. We include 42 RCTs (7935 women). The aromatase inhibitor letrozole was used in all studies. Letrozole compared to clomiphene citrate (CC) with or without adjuncts followed by timed intercourse Live birth rates were higher with letrozole (with or without adjuncts) compared to clomiphene citrate (with our without adjuncts) followed by timed intercourse (OR 1.68, 95% CI 1.42 to 1.99; 2954 participants; 13 studies; I2 = 0%; number needed to treat for an additional beneficial outcome (NNTB) = 10; moderate‐quality evidence). There is high‐quality evidence that OHSS rates are similar with letrozole or clomiphene citrate (0.5% in both arms: risk difference (RD) −0.00, 95% CI −0.01 to 0.00; 2536 participants; 12 studies; I2 = 0%; high‐quality evidence). There is evidence for a higher pregnancy rate in favour of letrozole (OR 1.56, 95% CI 1.37 to 1.78; 4629 participants; 25 studies; I2 = 1%; NNTB = 10; moderate‐quality evidence). There is little or no difference between treatment groups in the rate of miscarriage by pregnancy (20% with CC versus 19% with letrozole; OR 0.94, 95% CI 0.70 to 1.26; 1210 participants; 18 studies; I2 = 0%; high‐quality evidence) and multiple pregnancy rate (1.7% with CC versus 1.3% with letrozole; OR 0.69, 95% CI 0.41 to 1.16; 3579 participants; 17 studies; I2 = 0%; high‐quality evidence). However, a funnel plot showed mild asymmetry, indicating that some studies in favour of clomiphene might be missing. Letrozole compared to laparoscopic ovarian drilling There is low‐quality evidence that live birth rates are similar with letrozole or laparoscopic ovarian drilling (OR 1.38, 95% CI 0.95 to 2.02; 548 participants; 3 studies; I2 = 23%; low‐quality evidence). There is insufficient evidence for a difference in OHSS rates (RD 0.00, 95% CI −0.01 to 0.01; 260 participants; 1 study; low‐quality evidence). There is low‐quality evidence that pregnancy rates are similar (OR 1.28, 95% CI 0.94 to 1.74; 774 participants; 5 studies; I2 = 0%; moderate‐quality evidence). There is insufficient evidence for a difference in miscarriage rate by pregnancy (OR 0.66, 95% CI 0.30 to 1.43; 240 participants; 5 studies; I2 = 0%; moderate‐quality evidence), or multiple pregnancies (OR 3.00, 95% CI 0.12 to 74.90; 548 participants; 3 studies; I2 = 0%; low‐quality evidence). Additional comparisons were made for Letrozole versus placebo, Selective oestrogen receptor modulators (SERMS) followed by intrauterine insemination (IUI), follicle stimulating hormone (FSH), Anastrozole, as well as dosage and administration protocols. There is insufficient evidence for a difference in either group of treatment due to a limited number of studies. Hence more research is necessary. Authors' conclusions Letrozole appears to improve live birth and pregnancy rates in subfertile women with anovulatory polycystic ovary syndrome, compared to clomiphene citrate. There is high‐quality evidence that OHSS rates are similar with letrozole or clomiphene citrate. There is high‐quality evidence of no difference in miscarriage rates or multiple pregnancy rates. There is low‐quality evidence of no difference in live birth and pregnancy rates between letrozole and laparoscopic ovarian drilling, although there were few relevant studies. For the 2018 update, we added good‐quality trials, upgrading the quality of the evidence.

Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a coreceptor for growth factors and chemokines and is a molecular marker associated with epithelial-mesenchymal transition during development and carcinogenesis. Resistance of Syndecan-1-deficient mice to experimentally-induced tumorigenesis has been linked to altered Wnt-responsive precursor cell pools, suggesting a potential role of Syndecan-1 in breast cancer cell stem function. However, the precise molecular mechanism is still elusive. Here, we decipher the functional impact of Syndecan-1 knockdown using RNA interference on the breast cancer stem cell phenotype of human triple-negative MDA-MB-231 and hormone receptor-positive MCF-7 cells in vitro employing an analytical flow cytometric approach. Successful Syndecan-1 siRNA knockdown was confirmed by flow cytometry. Side population measurement by Hoechst dye exclusion and Aldehyde dehydrogenase-1 activity revealed that Syndecan-1 knockdown in MDA-MB-231 cells significantly reduced putative cancer stem cell pools by 60% and 27%, respectively, compared to controls. In MCF-7 cells, Syndecan-1 depletion reduced the side population by 40% and Aldehyde dehydrogenase-1 by 50%, repectively. In MDA-MB-231 cells, the CD44(+)CD24(-/low) phenotype decreased significantly by 6% upon siRNA-mediated Syndecan-1 depletion. Intriguingly, IL-6, its receptor sIL-6R, and the chemokine CCL20, implicated in regulating stemness-associated pathways, were downregulated by >40% in Syndecan-1-silenced MDA-MB-231 cells, which showed a dysregulated response to IL-6-induced shifts in E-cadherin and vimentin expression. Furthermore, activation of STAT-3 and NFkB transcription factors and expression of a coreceptor for Wnt signaling, LRP-6, were reduced by >45% in Syndecan-1-depleted cells compared to controls. At the functional level, Syndecan-1 siRNA reduced the formation of spheres and cysts in MCF-7 cells grown in suspension culture. Our study demonstrates the viability of flow cytometric approaches in analyzing cancer stem cell function. As Syndecan-1 modulates the cancer stem cell phenotype via regulation of the Wnt and IL-6/STAT3 signaling pathways, it emerges as a promising novel target for therapeutic approaches.

Endometrial cell clones derived from in vitro cultured purified stromal cells obtained by endometrial biopsy display characteristic stem cell features, including clonality; long-term culturing properties; multilineage differentiation potential; expression of CD146, CD105, CD90, CD73, MSI1, NOTCH1, and SOX2; and absence of CD34 and CD14 expression. We conclude that adult stem cells are present in endometrial biopsies performed in a routine clinical setting, offering new large-scale approaches for future research, diagnostics, and therapies involving adult stem cells. Endometrial cell clones derived from in vitro cultured purified stromal cells obtained by endometrial biopsy display characteristic stem cell features, including clonality; long-term culturing properties; multilineage differentiation potential; expression of CD146, CD105, CD90, CD73, MSI1, NOTCH1, and SOX2; and absence of CD34 and CD14 expression. We conclude that adult stem cells are present in endometrial biopsies performed in a routine clinical setting, offering new large-scale approaches for future research, diagnostics, and therapies involving adult stem cells. Pathologic conditions involving the endometrium such as endometriosis, adenomyosis, and endometrial carcinoma have been attributed to dysregulated stem cell (SC) function (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar, 2Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (255) Google Scholar, 3Gargett C.E. Uterine stem cells: what is the evidence?.Hum Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (268) Google Scholar, 4Du H. Taylor H.S. Stem cells and female reproduction.Reprod Sci. 2009; 16: 126-139Crossref PubMed Scopus (59) Google Scholar, 5Götte M. Endometrial cells get side-tracked: side population cells promote epithelial-mesenchymal transition in endometrial carcinoma.Am J Pathol. 2010; 176: 25-28Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Adult SCs have been isolated from menstrual blood (6Hida N. Nishiyama N. Miyoshi S. Kira S. Segawa K. Uyama T. et al.Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells.Stem Cells. 2008; 26: 1695-1704Crossref PubMed Scopus (258) Google Scholar, 7Patel A.N. Park E. Kuzman M. Benetti F. Silva F.J. Allickson J.G. Multipotent menstrual blood stromal stem cells: isolation, characterization, and differentiation.Cell Trans. 2008; 17: 303-311Crossref PubMed Scopus (276) Google Scholar), hysterectomy-derived endometrium (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar), and first-trimester decidua (9Dimitrov R. Kyurkchiev D. Timeva T. Yunakova M. Stamenova M. Shterev A. et al.First-trimester human decidua contains a population of mesenchymal stem cells.Fertil Steril. 2010; 93: 210-219Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), being characterized by SC marker gene expression (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar, 8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar, 10Matthai C. Horvat R. Noe M. Nagele F. Radjabi A. van Trotsenburg M. et al.Oct-4 expression in human endometrium.Mol Hum Reprod. 2006; 12: 7-10Crossref PubMed Scopus (92) Google Scholar, 11Wolf M. Kiesel L. Götte M. Endometrial stem cells. Potential relevance for the pathogenesis of endometriosis?.Gynakol Endokrinol. 2009; 7: 185-189Crossref Scopus (7) Google Scholar) and by their multilineage differentiation potential (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar, 12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar, 13Wolff E.F. Wolff A.B. Hongling Du Taylor H.S. Demonstration of multipotent stem cells in the adult human endometrium by in vitro chondrogenesis.Reprod Sci. 2007; 14: 524-533Crossref PubMed Scopus (119) Google Scholar). Large-scale studies of endometrial SCs are hampered by the limited availability of hysterectomy tissue. Endometrial biopsies may represent a viable alternative, as they are less invasive, preserve the uterus, and its function and are routinely performed for diagnostic purposes. Endometrial SCs may preferably cluster in the deeper layer of the basalis, constituting a reservoir for monthly regeneration of the endometrium (3Gargett C.E. Uterine stem cells: what is the evidence?.Hum Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (268) Google Scholar). However, recent data indicate that putative endometrial SCs may also reside in the superficial layers accessible by endometrial biopsy (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar). In this study, we characterize adult SCs in human endometrium obtained by routine biopsy in an IVF setting by their functional properties, including serial clonal passaging, long-term culturing properties, SC marker expression, and multilineage differentiation potential. In a university-based reproductive medicine unit, 36 female patients undergoing pretreatment diagnostics including endometrial biopsy were included in the study, which was carried out according to the principles of the Helsinki Convention and approved by the local ethics commission (no. 2008-223-f-S). All subjects gave written informed consent. Transcervical endometrial biopsies were taken on cycle days 22–24 as part of the diagnostic routine with a Probet catheter (Gynemed, Lensahn, Germany) (14Revel A. Multitasking human endometrium: a review of endometrial biopsy as a diagnostic tool, therapeutic applications, and a source of adult stem cells.Obstet Gynecol Surv. 2009; 64: 249-257Crossref PubMed Scopus (18) Google Scholar). One part of the samples was analyzed by the pathologist applying established criteria (15Noyes R.W. Hertig A.T. Rock J. Dating the endometrial biopsy.Fertil Steril. 1950; 1: 3-25Crossref Google Scholar), whereas the remaining part was transferred in HEPES-buffered Dulbecco’s modified essential medium (DMEM)/F-12 (Invitrogen, Carlsbad, CA). Endometrial stromal cells were isolated as described elsewhere (16Schwab K.E. Chan R.W. Gargett C.E. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle.Fertil Steril. 2005; 84: 1124-1130Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Enzymatically dissociated stromal cells were negatively selected using Dynabeads (Dynal, Oslo, Norway) specific for epithelial cells (BerEP4) and leukocytes (CD45) (17Chan R.W. Schwab K.E. Gargett C.E. Clonogenicity of human endometrial epithelial and stromal cells.Biol Reprod. 2004; 70: 1738-1750Crossref PubMed Scopus (477) Google Scholar). After erythrocyte lysis, cells were resuspended in DMEM/F-12/10% fetal calf serum (FCS) and cultured on fibronectin-coated 60-mm Petri dishes (BD Biosciences, Bedford, MA) at a density of 630 cells/3 mL in a humidified incubator (37°C/5% CO2). To determine cloning efficiency (CE) (16Schwab K.E. Chan R.W. Gargett C.E. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle.Fertil Steril. 2005; 84: 1124-1130Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), cells were cultured for 15 days, changing culture media every 6–7 days. Ten percent formaldehyde-fixed cells were stained with Crystal violet. Cell clusters were considered as colonies when they contained >50 cells. CE was defined as number of colonies/number of cells seeded × 100%. For serial passaging, a part of the cultures was used to determine CE, whereas the remaining part was passaged every 7–10 days upon reaching 80%–100% confluence for additional analyses. Expression of CD146 (MCAM), NOTCH1, Musashi-1 (MSI1), and SOX2 relative to 18S rRNA was analyzed by quantitative polymerase chain reaction (qPCR) as described elsewhere (18Götte M. Kalkhake K. Ploeger S. Kiesel L. Stute P. Effect of testosterone on E1S-sulfatase activity in non-malignant and cancerous breast cells in vitro.J Steroid Biochem Mol Biol. 2009; 117: 168-175Crossref PubMed Scopus (5) Google Scholar, 19Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (116613) Google Scholar) using TaqMan gene expression systems hs00174838_m1, hs00413187_m1, hs0015929_m1, hs00415716_m1, and hs99999901 s1 (ABI, Darmstadt, Germany) on three or more replicates. For flow-cytometric analysis, cells were trypsinized, washed, and resuspended in phosphate-buffered saline/2% bovine serum albumin. Fifty microliters of cells (1 × 106/mL) were incubated with fluorochrome-labeled monoclonal antibodies CD73-PE, IgG1κ isotype control (BD-Pharmingen, Heidelberg, Germany), CD105-PE, CD146-PC5, CD90-PC5, IgG2a-PC5 isotype control, CD45-PC7, CD34-PC7, CD14-PC7, IgG1-PC7 isotype control (Beckman Coulter, Krefeld, Germany) for 30 minutes at 20°C. Samples were diluted with 1 mL ISOTON II (Beckman Coulter) and analyzed in a Beckman Coulter FC500 flow-cytometer. Osteogenic differentiation of clonal endometrial stromal cells (n = 6 cultures) and of the pluripotent embryonal carcinoma cell line NTera-2 (20Andrews P.W. Damjanov I. Simon D. Banting G.S. Carlin C. Dracopoli N.C. et al.Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro.Lab Invest. 1984; 50: 147-162PubMed Google Scholar) was performed as described (21Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar). For 10 days before alkaline phosphatase activity staining using BCIP/NBT substrate (Sigma, Munich, Germany), 4.5 × 104 cells/1.5 mL were cultured on 35-mm cell culture dishes in NH-OsteoDiff Medium (Miltenyi, Bergisch-Gladbach, Germany). For adipogenic differentiation (21Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar), 7.5 × 104 cells/1.5 mL (n = 3) were cultured on 35-mm cell culture dishes in NH-AdipoDiff Medium (Miltenyi) for 21 days before staining with 0.5% Oil Red-O (Sigma). Induction media were changed every 3 days. Reverse transcription (RT)-PCR for PPARγ2 was performed as described (12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar). For chondrogenic differentiation (n = 2) (21Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar), 20,000 cells/200 μL were cultured in NH-ChondroDiff Medium (Miltenyi) for 24 days in 96-well plates before staining with 0.1 mg/mL Alcian Blue (Sigma). All negative control cells were cultured in DMEM/5% FCS. Conventional chromosome analysis was performed on long-term cultures analyzing 10 metaphase spreads at a 450-band level. Data are presented as mean ± SD. Unpaired t-tests were used to compare the significance between two groups. Data for cell yield were log transformed before t-test analysis. P<.05 was considered statistically significant. We obtained biopsies of 36 patients (mean age, 34.42 ± 5.10 years), of which 26 samples were secretory and 10 were proliferative. After plating immunopurified endometrial stroma cells at clonal density, clones emerged after 15 days (Fig. 1A ). Similar to hysterectomy-derived preparations (16Schwab K.E. Chan R.W. Gargett C.E. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle.Fertil Steril. 2005; 84: 1124-1130Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17Chan R.W. Schwab K.E. Gargett C.E. Clonogenicity of human endometrial epithelial and stromal cells.Biol Reprod. 2004; 70: 1738-1750Crossref PubMed Scopus (477) Google Scholar), large colonies containing a dense center of tightly packed cells with an overall swirly appearance (Fig. 1B) and small colonies of loosely packed cells (Fig. 1C) emerged. CE was 0.795% and increased to 4.329% after a second round of cloning (Fig. 1K), suggesting enrichment of SC activity. Among 16 independent clonal cultures that were passaged 12 times before cryoconservation, four cultures could be cultivated for >12 months and >30 serial passages without showing chromosomal aberrations (Fig. 1J). Endometrial cell clones were differentiated into multiple lineages (20Andrews P.W. Damjanov I. Simon D. Banting G.S. Carlin C. Dracopoli N.C. et al.Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro.Lab Invest. 1984; 50: 147-162PubMed Google Scholar, 21Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar). A subfraction of osteogenically induced clonal cells displayed alkaline phosphatase activity and a cuboidal morphology, demonstrating successful differentiation (Fig. 1D–1F). Successful formation of lipid vacuoles as a readout of adipocyte formation (22Okano H. Kawahara H. Toriya M. Nakao K. Shibata S. Imai T. Function of RNA-binding protein Musashi-1 in stem cells.Exp Cell Res. 2005; 306: 349-356Crossref PubMed Scopus (307) Google Scholar) was demonstrated by Oil Red-O staining and independently confirmed by PCR analysis for PPARγ2 (Fig. 1G–1I). Chondrogenic induction (20Andrews P.W. Damjanov I. Simon D. Banting G.S. Carlin C. Dracopoli N.C. et al.Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro.Lab Invest. 1984; 50: 147-162PubMed Google Scholar) resulted in the formation of comparably small (25–35 μm) Alcian Blue–positive cartilage-like nodular aggregates (not shown). We can only speculate whether these structures represent early stages of chondrogenic differentiation, as the formation of larger aggregates would be expected upon successful differentiation of mesenchymal SCs (MSCs) into chondrocytes. Expression of four adult SC markers and one pluripotency-associated SC marker was confirmed by qPCR (Fig. 1L–1N). Conforming with immunohistochemical data (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar), MSI1 and NOTCH1, which maintain SCs in an undifferentiated state (22Okano H. Kawahara H. Toriya M. Nakao K. Shibata S. Imai T. Function of RNA-binding protein Musashi-1 in stem cells.Exp Cell Res. 2005; 306: 349-356Crossref PubMed Scopus (307) Google Scholar), were expressed in clonal endometrial cells (Fig. 1L). Similar to hysterectomy-derived endometrium (12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar), CD146 was expressed in endometrial biopsy–derived cells. Expression of SOX2 (23Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14451) Google Scholar) was considerably lower compared with adult SC markers (Fig. 1M). A second round of culturing at clonal density resulted in enrichment of CD146, whereas MSI1 expression decreased (Fig. 1N). Flow-cytometric analysis of long-term clonal cultures of passages 18–34 revealed high expression of the MSC markers CD90 (98.45%, SD = 0.6, n = 2), CD73 (96.2%, SD = 2.3, n = 4), CD105 (96.8%, SD = 1.8, n = 2), and CD146 (75.3%, SD = 20.3, n = 4; Fig. 1O). Expression of CD34 (2.45%, SD = 0.2, n = 2) and CD14 (2.6%, SD = 1.4, n = 2) was at the limit of detection. Of two samples investigated for CD45 expression, one sample showed 12.3% CD45-positive cells, whereas a second sample contained 71.6% CD45-positive cells. Our study demonstrates the existence of MSC-like cells in human endometrium obtained by transcervical endometrial biopsy. Possibly owing to underrepresentation of the basalis layer, the CE in biopsy-derived endometrial stromal cell clones was slightly lower compared with hysterectomy-derived clones (0.8% vs. 1.1%–1.6%) (17Chan R.W. Schwab K.E. Gargett C.E. Clonogenicity of human endometrial epithelial and stromal cells.Biol Reprod. 2004; 70: 1738-1750Crossref PubMed Scopus (477) Google Scholar). In accordance with Schwab et al., no difference was observed between secretory and proliferative samples (16Schwab K.E. Chan R.W. Gargett C.E. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle.Fertil Steril. 2005; 84: 1124-1130Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Furthermore, when serum P as a continuous marker of secretory transition was evaluated, no correlation was observed with PCR markers (not shown). Similar to hysterectomy-derived samples (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar), CE increased significantly after the second round of cloning. Similarly (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar, 12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar), we demonstrated high expression of CD146 and a coexpression of the MSC markers CD73, CD105, and CD90 (24Dominici M. Le Blanc K. Mueller I. Slaper-Cortenbach I. Marini F. Krause D. et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.Cytotherapy. 2006; 8: 315-317Abstract Full Text Full Text PDF PubMed Scopus (11882) Google Scholar) in long-term cultures of serially passaged clones. Our cells were negative for markers of endothelial and primitive hematopoietic cells, monocytes, and macrophages. Our study therefore adds to the evidence for the presence of MSC-like cells in the human endometrium: Gargett and coworkers (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar, 12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar) could previously demonstrate that CD146+PDGFRß+MSC-like cells are located perivascularly in the functional and basal layers, and Hida et al. (6Hida N. Nishiyama N. Miyoshi S. Kira S. Segawa K. Uyama T. et al.Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells.Stem Cells. 2008; 26: 1695-1704Crossref PubMed Scopus (258) Google Scholar) could detect CD29+/CD105+/CD34−/CD45−MSC-like cells in menstrual blood. Since we immunodepleted CD45-positive cells during the isolation procedure, it is not fully clear whether flow-cytometric detection of CD45 in a subpopulation of our cultures indicates inefficient immunodepletion or a secondary reexpression induced by long-term culturing, possibly indicating a bone marrow–derived origin of our cells (2Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (255) Google Scholar, 4Du H. Taylor H.S. Stem cells and female reproduction.Reprod Sci. 2009; 16: 126-139Crossref PubMed Scopus (59) Google Scholar). Inclusion of analytical steps monitoring CD45 expression in early culture passages may be a reasonable addition to future isolation protocols. Quantitative PCR revealed similar CD146, MSI1, and NOTCH1 expression levels (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar). The finding that CD146 expression increased upon serial passaging, while MSI1 expression dropped, may indicate that these markers are expressed in different subpopulations. Specific growth factor supplementation or extracellular matrices may be required for propagation of MSI1-expressing cells. Detection of SOX2 is in accordance with endometrial expression of pluripotency-associated transcription factors such as OCT4 (10Matthai C. Horvat R. Noe M. Nagele F. Radjabi A. van Trotsenburg M. et al.Oct-4 expression in human endometrium.Mol Hum Reprod. 2006; 12: 7-10Crossref PubMed Scopus (92) Google Scholar) and NANOG (11Wolf M. Kiesel L. Götte M. Endometrial stem cells. Potential relevance for the pathogenesis of endometriosis?.Gynakol Endokrinol. 2009; 7: 185-189Crossref Scopus (7) Google Scholar), possibly suggesting the existence of different endometrial SC pools characterized by adult SC markers (CD146, MSI1, NOTCH1) or by an embryonic SC-like signature (SOX2, OCT4, NANOG), respectively. Our study provides novel evidence that routine endometrial biopsy is suitable to obtain MSC-like cells. Because the procedure is easy and performed routinely, a higher number of tissue samples can be obtained compared with hysterectomy. Since integrity and function of the organ is preserved, promising research strategies elucidating endometrial SC function during the female reproductive period will be possible. In the future, SCs derived by endometrial biopsy could provide an easily available source for the generation of induced pluripotent SCs. We thank Birgit Pers for expert technical assistance and Dr. Tobias Cantz, Dr. Andree Zibert, and Ramsi Siaj for helpful discussions.

Syndecan‐1 is a cell surface heparan sulfate proteoglycan with various biological functions relevant to tumor progression and inflammation, including cell–cell adhesion, cell–matrix interaction, and cytokine signaling driving cell proliferation and motility. Syndecan‐1 is a prognostic factor in breast cancer, and has a predicitive value for neodadjuvant chemotherapy. It is still poorly understood how syndecan‐1 integrates matrix‐dependent and cytokine‐dependent signaling processes in the tumor microenvironment. Here, we evaluated the potential role of syndecan‐1 in modulating matrix‐dependent breast cancer cell migration in the presence of interleukin‐6, and its potential involvement in resistance to irradiation in vitro . MDA ‐ MB ‐231 breast cancer cells were transiently transfected with syndecan‐1 small interfering RNA or control reagents, and this was followed by stimulation with interleukin‐6 or irradiation. Cellular responses were monitored by adhesion, migration and colony formation assays, as well as analysis of cell signaling. Syndecan‐1 depletion increased cell adhesion to fibronectin. Increased migration on fibronectin was significantly suppressed by interleukin‐6, and GRGDSP peptides inhibited both adhesion and migration. Interleukin‐6‐induced activation of focal adhesion kinase and reduction of constitutive nuclear factor kappaB signaling were decreased in syndecan‐1‐deficient cells. Focal adhesion kinase hyperactivation in syndecan‐1‐depleted cells was associated with dramatically reduced radiation sensitivity. We conclude that loss of syndecan‐1 leads to enhanced activation of β 1 ‐integrins and focal adhesion kinase, thus increasing breast cancer cell adhesion, migration, and resistance to irradiation. Syndecan‐1 deficiency also attenuates the modulatory effect of the inflammatory microenvironment constituent interleukin‐6 on cancer cell migration.

Endometriosis is a common disease but, due to the wide spectrum of symptoms, diagnosis can be delayed 8–12 years. Laparoscopy is nowadays the gold standard for diagnosis. A less invasive method could shorten the time to diagnosis. The aim of this review is to systematically summarize the literature regarding possible less invasive methods for the diagnosis of endometriosis. Electronic databases, including MEDLINE/PubMed, Cochrane, and Google Scholar, were searched to identify relevant studies; 53 publications contributed to this review. Low invasive tests including imaging, genetic tests, biomarkers, or miRNAs could be the key for establishing a less invasive diagnosis of endometriosis. The findings generally support that different methods can differently contribute to the diagnosis, also depending on the type of endometriosis. For example, transvaginal ultrasound has a sensitivity of 93% and a specificity of 96% in the diagnosis of endometrioma, while superficial/peritoneal endometriosis cannot be detected with imaging processes. Although several non-invasive tests including imaging, genetic tests, biomarkers, or miRNAs show promising diagnostic potential, further research is required before they can be recommended in routine clinical care. The combination of low invasive tests may be the solution to a reliable low invasive diagnosis of endometriosis.

MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR-142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of WASL (Wiskott-Aldrich syndrome-like, protein: N-WASP), Integrin-αV, RAC1, and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased Matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a change in cell morphology as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3'-untranslated region of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNA-dependent invasion phenotype. While knockdown of WASL significantly reduced the number of membrane protrusions compared to controls, knockdown of ITGAV resulted in a decreased cell volume, indicating differential contributions of these factors to the miR-142-3p-induced phenotype. Our data identify WASL, ITGAV and several additional cytoskeleton-associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer.

Effectively targeting cancer stem cells, a subpopulation of tumorigenic, aggressive, and radioresistant cells, holds therapeutic promise. However, the effects of the microRNA miR-142-3p, a small endogenous regulator of gene expression on breast cancer stem cells, have not been investigated. This study identifies the influence of miR-142-3p on mammary stemness properties and breast cancer radioresistance to establish its role in this setting. miR-142-3p precursor transfection was performed in MDA-MB-468, HCC1806, and MCF-7 cells, and stem cell markers CD44, CD133, ALDH1 activity and mammosphere formation were measured. β-catenin, the canonical wnt signaling effector protein, was quantified by Western blots and cell fluorescence assays both in miR-142-3p–overexpressing and anti–miR-142-3p–treated cells. Radiation response was investigated by colony formation assays. Levels of BRCA1, BRCA2, and Bod1 in miR-142-3p–overexpressing cells as well as expression of miR-142-3p, Bod1, KLF4, and Oct4 in sorted CD44 + /CD24 –/low cells were determined by quantitative polymerase chain reaction. miR-142-3p overexpression resulted in a strong decline in breast cancer stem cell characteristics with a decrease in CD44, CD133, ALDH1, Bod1, BRCA2, and mammosphere formation as well as reduced survival after irradiation. miR-142-3p expression was strongly reduced in sorted CD44 + /CD24 –/low stem cells, while Bod1, Oct4, and KLF4 were overexpressed. β-catenin levels strongly decreased after miR-142-3p overexpression, but not after anti–miR-142-3p treatment. We conclude that miR-142-3p downregulates cancer stem cell characteristics and radioresistance in breast cancer, mediated by a reduced role of β-catenin in miR-142-3p–overexpressing cells. miR-142-3p might therefore help to target cancer stem cells.

Endometriosis is characterized by growth of endometrial tissue at ectopic locations. Down-regulation of microRNA miR-200b is observed in endometriosis and malignant disease, driving tumour cells towards an invasive state by enhancing epithelial-to-mesenchymal transition (EMT). miR-200b up-regulation may inhibit EMT and invasive growth in endometriosis. To study its functional impact on the immortalized endometriotic cell line 12Z, the stromal cell line ST-T1b, and primary endometriotic stroma cells, a transient transfection approach with microRNA precursors was employed. Expression of bioinformatically predicted targets of miR-200b was analysed by qPCR. The cellular phenotype was monitored by Matrigel invasion assays, digital-holographic video microscopy and flow cytometry. qPCR revealed significant down-regulation of ZEB1 (P < 0.05) and ZEB2 (P < 0.01) and an increase in E-cadherin (P < 0.01). miR-200b overexpression decreased invasiveness (P < 0.0001) and cell motility (P < 0.05). In contrast, cell proliferation (P < 0.0001) and the stemness-associated side population phenotype (P < 0.01) were enhanced following miR-200b transfection. These properties were possibly due to up-regulation of the pluripotency-associated transcription factor KLF4 (P < 0.05) and require attention when considering therapeutic strategies. In conclusion, up-regulation of miR-200b reverts EMT, emerging as a potential therapeutic approach to inhibit endometriotic cell motility and invasiveness.

Dyslipidemias are common and increase the risk of cardiovascular disease. The menopause transition is associated with an atherogenic lipid profile, with an increase in the concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), apolipoprotein B (apoB) and potentially lipoprotein (a) [Lp(a)], and a decrease in the concentration of high-density lipoprotein cholesterol (HDL-C).The aim of this clinical guide is to provide an evidence-based approach to management of menopausal symptoms and dyslipidemia in postmenopausal women. The guide evaluates the effects on the lipid profile both of menopausal hormone therapy and of non-estrogen-based treatments for menopausal symptoms.Literature review and consensus of expert opinion.Initial management depends on whether the dyslipidemia is primary or secondary. An assessment of the 10-year risk of fatal cardiovascular disease, based on the Systematic Coronary Risk Estimation (SCORE) system, should be used to set the optimal LDL-C target. Dietary changes and pharmacological management of dyslipidemias should be tailored to the type of dyslipidemia, with statins constituting the mainstay of treatment. With regard to menopausal hormone therapy, systemic estrogens induce a dose-dependent reduction in TC, LDL-C and Lp(a), as well as an increase in HDL-C concentrations; these effects are more prominent with oral administration. Transdermal rather than oral estrogens should be used in women with hypertriglyceridemia. Micronized progesterone or dydrogesterone are the preferred progestogens due to their neutral effect on the lipid profile. Tibolone may decrease TC, LDL-C, TG and Lp(a), but also HDL-C concentrations. Low-dose vaginal estrogen and ospemifene exert a favorable effect on the lipid profile, but data are scant regarding dehydroepiandrosterone (DHEA). Non-estrogen-based therapies, such as fluoxetine and citalopram, exert a more favorable effect on the lipid profile than do sertraline, paroxetine and venlafaxine. Non-oral testosterone, used for the treatment of hypoactive sexual desire disorder/dysfunction, has little or no effect on the lipid profile.

Smoking by one or both partners can adversely affect IVF outcome. We investigated whether smoking may also play a role in the success rate of intracytoplasmic sperm injection (ICSI), in which initial steps of fertilization are bypassed.Three hundred one couples (ICSI: 153, IVF: 148) participated in 415 treatment cycles (ICSI: 202, IVF: 213). One hundred thirty-nine men were habitual smokers (ICSI: 71, IVF: 68). Seventy-seven women were smokers (ICSI: 41, IVF: 36). Multiple nominal regression analyses of various steps of assisted reproduction included smoking status, age, semen parameters, and number of embryos transferred.Reproductive and andrology unit of the university.Three hundred one couples seeking fertility treatment.Assisted reproduction by in vitro fertilization (IVF) or ICSI.Clinical pregnancy.Intracytoplasmic sperm injection success (clinical pregnancy) in women with smoking male partners was 22% and was 38% with nonsmoking partners. Similar results were seen for IVF, with 18% vs. 32%. Multinominal logistic regression analysis revealed smoking in men to be a significant predictor of ICSI outcome, along with female age and the number of embryos transferred, whereas clinical pregnancies after IVF were dependent on smoking in men, number of embryos transferred, sperm motility, and female age. Female smoking influenced the number of oocytes retrieved and the fertilization rate of oocytes in IVF but not in ICSI. The odds ratio for failure of ICSI for male smokers in comparison to male nonsmokers was 2.95 (IVF: 2.65).Smoking by males decreases the success rates of assisted reproduction procedures, not only in IVF, but also in ICSI. Apart from putative adverse effects during fertilization, altered DNA in spermatozoa might hamper development of the embryo.

Endometriotic lesions secrete chemokines that recruit immune cells into the peritoneal cavity. The accumulation of these immune cells, especially activated macrophages and T lymphocytes, is thought to mediate inflammatory symptoms associated with endometriosis. Previous studies have demonstrated that RANTES (regulated on activation, normal T cell expressed and secreted) is synthesized by endometriotic stromal cells and circulates in peritoneal fluid, commensurate with the stage of endometriosis. In the current studies, we used the human monocytic cell line, U937, to assay chemotactic activity in cell culture conditioned media and peritoneal fluid from patients with endometriosis and normal controls. We demonstrated expression of the human RANTES receptors CCR-1 and CCR-5 in U937 cells and peritoneal macrophages. Over a range of 0-1000 pg/ml recombinant human RANTES had a direct, linear effect on monocyte migration. Conditioned media and peritoneal fluid induced dose-dependent effects on monocyte migration that were correlated with concentrations of immunoreactive RANTES (as measured by enzyme-linked immunosorbent assay) and the severity of endometriosis. Heat denaturation of the RANTES protein or addition of anti-human RANTES antibodies neutralized the chemoattractant effects of conditioned media and peritoneal fluid. RANTES stimulation of monocyte recruitment may be an important pathogenetic target for the treatment of infertility and pain associated with endometriosis.

The aim of this study was to investigate the expression of the protein tyrosine phosphatases (PTP) PRL-1, PRL-2, and PRL-3 in human breast cancer and to evaluate its clinical and prognostic significance. PRL-PTP mRNA expression was examined in malignant (n = 7) and nonmalignant (n = 7) cryoconserved breast tissue samples as well as in eight breast cancer cell lines by RT-PCR. Furthermore, protein expression of PRL-3 was analysed semiquantitatively by immunohistochemistry in ductal breast carcinoma in situ (n = 135) and invasive breast cancer (n = 147) by use of tissue microarray technology (TMA). In 24 lymph node-positive patients we selected the corresponding lymph node metastases for analysis of PRL-3 expression, and a validation set (n = 99) of invasive breast cancer samples was examined. Staining results were correlated with clinicopathological parameters and long-term follow-up. PRL-3 mRNA expression was significantly higher in malignant compared to benign breast tissue. For PRL-1 and PRL-2 expression no significant differences were observed. Staining of TMAs showed PRL-3 expression in 85.9% ductal carcinoma in situ and 75.5% invasive breast carcinomas. Analysis of survival parameters revealed a shorter disease-free survival (DFS) in patients with PRL-3-positive carcinomas, and in particular a significantly shorter DFS in nodal-positive patients with PRL-3 overexpressing tumours as compared to PRL-3-negative breast carcinomas (66+/-7 months (95% CI, 52-80) vs 97+/-9 months (95% CI, 79-115); P = 0.032). Moreover, we found a more frequent expression of PRL-3 in lymph node metastases as compared to the primary tumours (91.7 vs 66.7%; P = 0.033). Our results suggest that PRL-3 might serve as a novel prognostic factor in breast cancer, which may help to predict an adverse disease outcome.

Heparan sulphate proteoglycan syndecan-1 modulates cell proliferation, adhesion, migration and angiogenesis. It is a coreceptor for the hepatocyte growth factor receptor c-met, and its coexpression with E-cadherin is synchronously regulated during epithelial-mesenchymal transition. In breast cancer, changes in the expression of syndecan-1, E-cadherin and c-met correlate with poor prognosis. In this study we evaluated whether coexpression of these functionally linked prognostic markers constitutes an expression signature in ductal carcinoma in situ (DCIS) of the breast that may promote cell proliferation and (lymph)angiogenesis.Expression of syndecan-1, E-cadherin and c-met was detected immunohistochemically using a tissue microarray in tumour specimens from 200 DCIS patients. Results were correlated with the expression patterns of angiogenic and lymphangiogenic markers. Coexpression of the three prognostic markers was evaluated in human breast cancer cells by confocal immunofluorescence microscopy and RT-PCR.Coexpression and membrane colocalization of the three markers was confirmed in MCF-7 cells. E-cadherin expression decreased, and c-met expression increased progressively in more aggressive cell lines. Tissue microarray analysis revealed strong positive staining of tumour cells for syndecan-1 in 72%, E-cadherin in 67.8% and c-met in 48.6% of DCIS. E-cadherin expression was significantly associated with c-met and syndecan-1. Expression of c-met and syndecan-1 was significantly more frequent in the subgroup of patients with pure DCIS than in those with DCIS and a coexisting invasive carcinoma. Levels of c-met and syndecan-1 expression were associated with HER2 expression. Expression of c-met significantly correlated with expression of endothelin A and B receptors, vascular endothelial growth factor (VEGF)-A and fibroblast growth factor receptor-1, whereas E-cadherin expression correlated significantly with endothelin A receptor, VEGF-A and VEGF-C staining.Syndecan-1, E-cadherin and c-met constitute a marker signature associated with angiogenic and lymphangiogenic factors in DCIS. This coexpression may reflect a state of parallel activation of different signal transduction pathways, promoting tumour cell proliferation and angiogenesis. Our findings have implications for future therapeutic approaches in terms of a multiple target approach, which may be useful early in breast cancer progression.

The RNA-binding protein Musashi-1 has been proposed to maintain stem cell function during development and regenerative processes as a modulator of the Notch-1 signaling pathway. Musashi-1 expression is upregulated in endometrial carcinoma, however, its pathogenetic role in this tumor entity is unknown. Here we investigate the functional impact and mode of action of Musashi-1 on endometrial carcinoma cell behaviour in vitro. Aldehyde dehydrogenase-1 activity and side population (SP) measurement by Hoechst dye exclusion revealed that the Ishikawa endometrial carcinoma cell line contains a pool of putative cancer stem cells. Musashi-1 expression is 20.8-fold upregulated in SP+ compared to SP- and equally distributed between ALDH+ and ALDH- cell pools. siRNA-mediated knockdown of Musashi-1 mRNA expression lead to an altered expression of the signaling receptor Notch-1 and its downstream targets, the transcription factor Hes-1 and the cell cycle regulators p21(WAF1/CIP1) and cyclin B1, as determined by Western blotting and quantitative real-time PCR. Flow cytometric and ELISA analyses revealed that Musashi-1-mediated modulation of these factors exerted an antiproliferative effect on the cell cycle, and increased apoptosis in endometrial carcinoma cells. We conclude that Ishikawa cells contain a subpopulation of cells with stem cell-like properties. Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via the stemness-related factors Notch-1, Hes-1 and p21(WAF1/CIP1) , thus emerging as a novel future target for endometrial carcinoma therapy.

ObjectiveTo study the function of miR-145, known to be dysregulated in endometriosis, and to identify its target genes in an in vitro endometriosis model.DesignExperimental laboratory study.SettingUniversity medical centers.Patient(s)Primary endometrial stroma cells were derived from eutopic endometrium of three American Society for Reproductive Medicine stage III endometriosis patients and from ectopic lesions of four patients with deep infiltrating endometriosis.Intervention(s)The human endometriotic cell line 12Z and primary eutopic and ectopic endometrial stroma cells were transiently transfected with miR-145 precursors or anti–miR-145 inhibitors and investigated for posttranscriptional regulation of predicted target genes and changes in cell behavior.Main Outcome Measure(s)Predicted target expression was measured by quantitative reverse transcription–polymerase chain reaction and Western blotting, and altered cell behavior was monitored by cell proliferation assays. The 12Z cells were additionally investigated by Matrigel invasion assays, cell cycle analysis, side population analysis, and aldehyde dehydrogenase activity assays.Result(s)In all cells investigated, miR-145 overexpression inhibited cell proliferation and induced down-regulation of FASCIN-1, SOX2, and MSI2. In 12Z cells miR-145 upregulation increased Matrigel invasiveness and reduced side population and aldehyde dehydrogenase-1 activity. Additional down-regulated targets in 12Z cells included OCT4, KLF4, PODXL, JAM-A, and SERPINE1/PAI-1. ACTG2 and TAGLN were up-regulated upon pre–miR-145 transfection. JAM-A, FASCIN-1, and PAI-I down-regulation in 12Z cells were confirmed by Western blotting.Conclusion(s)miR-145 inhibits endometriotic cell proliferation, invasiveness, and stemness by targeting multiple pluripotency factors, cytoskeletal elements, and protease inhibitors. To study the function of miR-145, known to be dysregulated in endometriosis, and to identify its target genes in an in vitro endometriosis model. Experimental laboratory study. University medical centers. Primary endometrial stroma cells were derived from eutopic endometrium of three American Society for Reproductive Medicine stage III endometriosis patients and from ectopic lesions of four patients with deep infiltrating endometriosis. The human endometriotic cell line 12Z and primary eutopic and ectopic endometrial stroma cells were transiently transfected with miR-145 precursors or anti–miR-145 inhibitors and investigated for posttranscriptional regulation of predicted target genes and changes in cell behavior. Predicted target expression was measured by quantitative reverse transcription–polymerase chain reaction and Western blotting, and altered cell behavior was monitored by cell proliferation assays. The 12Z cells were additionally investigated by Matrigel invasion assays, cell cycle analysis, side population analysis, and aldehyde dehydrogenase activity assays. In all cells investigated, miR-145 overexpression inhibited cell proliferation and induced down-regulation of FASCIN-1, SOX2, and MSI2. In 12Z cells miR-145 upregulation increased Matrigel invasiveness and reduced side population and aldehyde dehydrogenase-1 activity. Additional down-regulated targets in 12Z cells included OCT4, KLF4, PODXL, JAM-A, and SERPINE1/PAI-1. ACTG2 and TAGLN were up-regulated upon pre–miR-145 transfection. JAM-A, FASCIN-1, and PAI-I down-regulation in 12Z cells were confirmed by Western blotting. miR-145 inhibits endometriotic cell proliferation, invasiveness, and stemness by targeting multiple pluripotency factors, cytoskeletal elements, and protease inhibitors.

Heparan sulfate 3‐ O ‐sulfotransferase 2 (HS3ST2), an enzyme mediating 3‐ O ‐sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co‐receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2 ‐expressing MDA‐MB‐231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real‐time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin‐11, E‐cadherin and CEACAM‐1 , while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 ‐expressing MCF‐7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS‐dependent basal and FGF2‐specific signaling through the constitutively active p44/42 MAPK pathway in MDA‐MB‐231 cells. Increased MAPK activation was accompanied by upregulation of ß‐catenin in MDA‐MB‐231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4‐regulated ion transporters and increased cytosolic acidification were observed in HS3ST2 ‐expressing MDA‐MB‐231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.

Abstract Aim The aim of this official guideline published by the German Society of Gynecology and Obstetrics (DGGG) and coordinated with the German Society of Urology (DGU) and the German Society of Reproductive Medicine (DGRM) is to provide consensus-based recommendations, obtained by evaluating the relevant literature, on counseling and fertility preservation for prepubertal girls and boys as well as patients of reproductive age. Statements and recommendations for girls and women are presented below. Statements or recommendations for boys and men are not the focus of this guideline. Methods This S2k guideline was developed at the suggestion of the guideline commission of the DGGG, DGU and DGRM and represents the structured consensus of representative members from various professional associations (n = 40). Recommendations The guideline provides recommendations on counseling and fertility preservation for women and girls which take account of the patientʼs personal circumstances, the planned oncologic therapy and the individual risk profile as well as the preferred approach for selected tumor entities.

Can the priorities for future research in infertility be identified?The top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction and ethics, access and organization of care for people with fertility problems were identified.Many fundamental questions regarding the prevention, management and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems.Potential research questions were collated from an initial international survey, a systematic review of clinical practice guidelines and Cochrane systematic reviews. A rationalized list of confirmed research uncertainties was prioritized in an interim international survey. Prioritized research uncertainties were discussed during a consensus development meeting. Using a formal consensus development method, the modified nominal group technique, diverse stakeholders identified the top 10 research priorities for each of the categories male infertility, female and unexplained infertility, medically assisted reproduction and ethics, access and organization of care.Healthcare professionals, people with fertility problems and others (healthcare funders, healthcare providers, healthcare regulators, research funding bodies and researchers) were brought together in an open and transparent process using formal consensus methods advocated by the James Lind Alliance.The initial survey was completed by 388 participants from 40 countries, and 423 potential research questions were submitted. Fourteen clinical practice guidelines and 162 Cochrane systematic reviews identified a further 236 potential research questions. A rationalized list of 231 confirmed research uncertainties was entered into an interim prioritization survey completed by 317 respondents from 43 countries. The top 10 research priorities for each of the four categories male infertility, female and unexplained infertility (including age-related infertility, ovarian cysts, uterine cavity abnormalities and tubal factor infertility), medically assisted reproduction (including ovarian stimulation, IUI and IVF) and ethics, access and organization of care were identified during a consensus development meeting involving 41 participants from 11 countries. These research priorities were diverse and seek answers to questions regarding prevention, treatment and the longer-term impact of infertility. They highlight the importance of pursuing research which has often been overlooked, including addressing the emotional and psychological impact of infertility, improving access to fertility treatment, particularly in lower resource settings and securing appropriate regulation. Addressing these priorities will require diverse research methodologies, including laboratory-based science, qualitative and quantitative research and population science.We used consensus development methods, which have inherent limitations, including the representativeness of the participant sample, methodological decisions informed by professional judgment and arbitrary consensus definitions.We anticipate that identified research priorities, developed to specifically highlight the most pressing clinical needs as perceived by healthcare professionals, people with fertility problems and others, will help research funding organizations and researchers to develop their future research agenda.The study was funded by the Auckland Medical Research Foundation, Catalyst Fund, Royal Society of New Zealand and Maurice and Phyllis Paykel Trust. G.D.A. reports research sponsorship from Abbott, personal fees from Abbott and LabCorp, a financial interest in Advanced Reproductive Care, committee membership of the FIGO Committee on Reproductive Medicine, International Committee for Monitoring Assisted Reproductive Technologies, International Federation of Fertility Societies and World Endometriosis Research Foundation, and research sponsorship of the International Committee for Monitoring Assisted Reproductive Technologies from Abbott and Ferring. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and editor for the Cochrane Gynaecology and Fertility Group. J.L.H.E. reports being the Editor Emeritus of Human Reproduction. A.W.H. reports research sponsorship from the Chief Scientist's Office, Ferring, Medical Research Council, National Institute for Health Research and Wellbeing of Women and consultancy fees from AbbVie, Ferring, Nordic Pharma and Roche Diagnostics. M.L.H. reports grants from Merck, grants from Myovant, grants from Bayer, outside the submitted work and ownership in Embrace Fertility, a private fertility company. N.P.J. reports research sponsorship from AbbVie and Myovant Sciences and consultancy fees from Guerbet, Myovant Sciences, Roche Diagnostics and Vifor Pharma. J.M.L.K. reports research sponsorship from Ferring and Theramex. R.S.L. reports consultancy fees from AbbVie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. B.W.M. reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. E.H.Y.N. reports research sponsorship from Merck. C.N. reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring and retains a financial interest in NexHand. J.S. reports being employed by a National Health Service fertility clinic, consultancy fees from Merck for educational events, sponsorship to attend a fertility conference from Ferring and being a clinical subeditor of Human Fertility. A.S. reports consultancy fees from Guerbet. J.W. reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. A.V. reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and the journal Reproduction. His employing institution has received payment from Human Fertilisation and Embryology Authority for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. N.L.V. reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the present work. All authors have completed the disclosure form.N/A.

Globally, 985 million women are aged 50 and over, leading to increasing concerns about chronic conditions such as cardiovascular disease, osteoporosis, dementia, and cognitive decline, which can adversely affect quality of life and independent living.To evaluate the evidence from observational studies and randomized trials on the effects of the Mediterranean diet on short- and long-term menopausal health: estrogen deficiency symptoms, cardiovascular disease, osteoporosis, cognitive and mental health, breast cancer, and all-cause mortality.Literature review and consensus of expert opinion.The Mediterranean diet is a non-restrictive dietary pattern common in the olive-growing areas of the Mediterranean basin. It may improve vasomotor symptoms, cardiovascular risk factors such as blood pressure, cholesterol and blood glucose levels, as well as mood and symptoms of depression. Long-term adherence may: improve cardiovascular risk and events, and death; improve bone mineral density; prevent cognitive decline; and reduce the risk of breast cancer and all-cause mortality.

Vulvovaginal atrophy (VVA) is a chronic condition caused by estrogen deficiency. It affects around 50% of postmenopausal women, reducing their general and sexual quality of life as well as the quality of their personal relationships.The aim of this clinical guide is to set out an individualized approach to the management of VVA with topical estrogens and non-hormonal preparations.Literature review and consensus of expert opinion.An individualized approach is required for the management of VVA. Topical low-dose estrogens are effective and also alleviate urinary incontinence and prevent recurrent urinary tract infections. Women should not be denied long-term use of topical estrogens as long as they feel that this treatment is of benefit to them, because the safety data are reassuring. Non-hormonal preparations (lubricants and moisturizers) should be the first-line treatment for VVA in women taking adjuvant endocrine therapies for cancers considered to be hormone-dependent. They can be used over the long term.

Study QuestionCan the priorities for future research in infertility be identified?Summary AnswerThe top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care for people with fertility problems were identified.What is Known AlreadyMany fundamental questions regarding the prevention, management, and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems.Study Design, Size, DurationPotential research questions were collated from an initial international survey, a systematic review of clinical practice guidelines, and Cochrane systematic reviews. A rationalized list of confirmed research uncertainties was prioritized in an interim international survey. Prioritized research uncertainties were discussed during a consensus development meeting. Using a formal consensus development method, the modified nominal group technique, diverse stakeholders identified the top 10 research priorities for each of the categories male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care.Participants/Materials, Setting, MethodsHealthcare professionals, people with fertility problems, and others (healthcare funders, healthcare providers, healthcare regulators, research funding bodies and researchers) were brought together in an open and transparent process using formal consensus methods advocated by the James Lind Alliance.Main Results and the Role of ChanceThe initial survey was completed by 388 participants from 40 countries, and 423 potential research questions were submitted. Fourteen clinical practice guidelines and 162 Cochrane systematic reviews identified a further 236 potential research questions. A rationalized list of 231 confirmed research uncertainties were entered into an interim prioritization survey completed by 317 respondents from 43 countries. The top 10 research priorities for each of the four categories male infertility, female and unexplained infertility (including age-related infertility, ovarian cysts, uterine cavity abnormalities, and tubal factor infertility), medically assisted reproduction (including ovarian stimulation, IUI, and IVF), and ethics, access, and organization of care, were identified during a consensus development meeting involving 41 participants from 11 countries. These research priorities were diverse and seek answers to questions regarding prevention, treatment, and the longer-term impact of infertility. They highlight the importance of pursuing research which has often been overlooked, including addressing the emotional and psychological impact of infertility, improving access to fertility treatment, particularly in lower resource settings, and securing appropriate regulation. Addressing these priorities will require diverse research methodologies, including laboratory-based science, qualitative and quantitative research, and population science.Limitations, Reasons for CautionWe used consensus development methods, which have inherent limitations, including the representativeness of the participant sample, methodological decisions informed by professional judgement, and arbitrary consensus definitions.Wider Implications of the FindingsWe anticipate that identified research priorities, developed to specifically highlight the most pressing clinical needs as perceived by healthcare professionals, people with fertility problems, and others, will help research funding organizations and researchers to develop their future research agenda.Study Funding/ Competing Interest(s)The study was funded by the Auckland Medical Research Foundation, Catalyst Fund, Royal Society of New Zealand, and Maurice and Phyllis Paykel Trust. Geoffrey Adamson reports research sponsorship from Abbott, personal fees from Abbott and LabCorp, a financial interest in Advanced Reproductive Care, committee membership of the FIGO Committee on Reproductive Medicine, International Committee for Monitoring Assisted Reproductive Technologies, International Federation of Fertility Societies, and World Endometriosis Research Foundation, and research sponsorship of the International Committee for Monitoring Assisted Reproductive Technologies from Abbott and Ferring. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and editor for the Cochrane Gynaecology and Fertility Group. Hans Evers reports being the Editor Emeritus of Human Reproduction. Andrew Horne reports research sponsorship from the Chief Scientist’s Office, Ferring, Medical Research Council, National Institute for Health Research, and Wellbeing of Women and consultancy fees from Abbvie, Ferring, Nordic Pharma, and Roche Diagnostics. M. Louise Hull reports grants from Merck, grants from Myovant, grants from Bayer, outside the submitted work and ownership in Embrace Fertility, a private fertility company. Neil Johnson reports research sponsorship from Abb-Vie and Myovant Sciences and consultancy fees from Guerbet, Myovant Sciences, Roche Diagnostics, and Vifor Pharma. José Knijnenburg reports research sponsorship from Ferring and Theramex. Richard Legro reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. Ben Mol reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. Ernest Ng reports research sponsorship from Merck. Craig Niederberger reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and retains a financial interest in NexHand. Jane Stewart reports being employed by a National Health Service fertility clinic, consultancy fees from Merck for educational events, sponsorship to attend a fertility conference from Ferring, and being a clinical subeditor of Human Fertility. Annika Strandell reports consultancy fees from Guerbet. Jack Wilkinson reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. Andy Vail reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and of the journal Reproduction. His employing institution has received payment from HFEA for his advice on review of research evidence to inform their ‘traffic light’ system for infertility treatment ‘add-ons'. Lan Vuong reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the present work. All authors have completed the disclosure form.Trial Registration NumberNot applicable. Can the priorities for future research in infertility be identified? The top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care for people with fertility problems were identified. Many fundamental questions regarding the prevention, management, and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems. Potential research questions were collated from an initial international survey, a systematic review of clinical practice guidelines, and Cochrane systematic reviews. A rationalized list of confirmed research uncertainties was prioritized in an interim international survey. Prioritized research uncertainties were discussed during a consensus development meeting. Using a formal consensus development method, the modified nominal group technique, diverse stakeholders identified the top 10 research priorities for each of the categories male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care. Healthcare professionals, people with fertility problems, and others (healthcare funders, healthcare providers, healthcare regulators, research funding bodies and researchers) were brought together in an open and transparent process using formal consensus methods advocated by the James Lind Alliance. The initial survey was completed by 388 participants from 40 countries, and 423 potential research questions were submitted. Fourteen clinical practice guidelines and 162 Cochrane systematic reviews identified a further 236 potential research questions. A rationalized list of 231 confirmed research uncertainties were entered into an interim prioritization survey completed by 317 respondents from 43 countries. The top 10 research priorities for each of the four categories male infertility, female and unexplained infertility (including age-related infertility, ovarian cysts, uterine cavity abnormalities, and tubal factor infertility), medically assisted reproduction (including ovarian stimulation, IUI, and IVF), and ethics, access, and organization of care, were identified during a consensus development meeting involving 41 participants from 11 countries. These research priorities were diverse and seek answers to questions regarding prevention, treatment, and the longer-term impact of infertility. They highlight the importance of pursuing research which has often been overlooked, including addressing the emotional and psychological impact of infertility, improving access to fertility treatment, particularly in lower resource settings, and securing appropriate regulation. Addressing these priorities will require diverse research methodologies, including laboratory-based science, qualitative and quantitative research, and population science. We used consensus development methods, which have inherent limitations, including the representativeness of the participant sample, methodological decisions informed by professional judgement, and arbitrary consensus definitions. We anticipate that identified research priorities, developed to specifically highlight the most pressing clinical needs as perceived by healthcare professionals, people with fertility problems, and others, will help research funding organizations and researchers to develop their future research agenda. The study was funded by the Auckland Medical Research Foundation, Catalyst Fund, Royal Society of New Zealand, and Maurice and Phyllis Paykel Trust. Geoffrey Adamson reports research sponsorship from Abbott, personal fees from Abbott and LabCorp, a financial interest in Advanced Reproductive Care, committee membership of the FIGO Committee on Reproductive Medicine, International Committee for Monitoring Assisted Reproductive Technologies, International Federation of Fertility Societies, and World Endometriosis Research Foundation, and research sponsorship of the International Committee for Monitoring Assisted Reproductive Technologies from Abbott and Ferring. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and editor for the Cochrane Gynaecology and Fertility Group. Hans Evers reports being the Editor Emeritus of Human Reproduction. Andrew Horne reports research sponsorship from the Chief Scientist’s Office, Ferring, Medical Research Council, National Institute for Health Research, and Wellbeing of Women and consultancy fees from Abbvie, Ferring, Nordic Pharma, and Roche Diagnostics. M. Louise Hull reports grants from Merck, grants from Myovant, grants from Bayer, outside the submitted work and ownership in Embrace Fertility, a private fertility company. Neil Johnson reports research sponsorship from Abb-Vie and Myovant Sciences and consultancy fees from Guerbet, Myovant Sciences, Roche Diagnostics, and Vifor Pharma. José Knijnenburg reports research sponsorship from Ferring and Theramex. Richard Legro reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. Ben Mol reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. Ernest Ng reports research sponsorship from Merck. Craig Niederberger reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and retains a financial interest in NexHand. Jane Stewart reports being employed by a National Health Service fertility clinic, consultancy fees from Merck for educational events, sponsorship to attend a fertility conference from Ferring, and being a clinical subeditor of Human Fertility. Annika Strandell reports consultancy fees from Guerbet. Jack Wilkinson reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. Andy Vail reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and of the journal Reproduction. His employing institution has received payment from HFEA for his advice on review of research evidence to inform their ‘traffic light’ system for infertility treatment ‘add-ons'. Lan Vuong reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the present work. All authors have completed the disclosure form.

Endometriosis is a painful gynecological condition characterized by ectopic growth of endometrial cells. Little is known about its pathogenesis, which is partially due to a lack of suitable experimental models. Here, we use endometrial stromal (St-T1b), primary endometriotic stromal, epithelial endometriotic (12Z) and co-culture (1:1 St-T1b:12Z) spheroids to mimic the architecture of endometrium, and either collagen I or Matrigel to model ectopic locations. Stromal spheroids, but not single cells, assumed coordinated directional migration followed by matrix remodeling of collagen I on day 5 or 7, resembling ectopic lesions. While generally a higher area fold increase of spheroids occurred on collagen I compared to Matrigel, directional migration was not observed in co-culture or in 12Z cells. The fold increase in area on collagen I was significantly reduced by MMP inhibition in stromal but not 12Z cells. Inhibiting ROCK signalling responsible for actomyosin contraction increased the fold increase of area and metabolic activity compared to untreated controls on Matrigel. The number of protrusions emanating from 12Z spheroids on Matrigel was decreased by microRNA miR-200b and increased by miR-145. This study demonstrates that spheroid assay is a promising pre-clinical tool that can be used to evaluate small molecule drugs and microRNA-based therapeutics for endometriosis.

Worldwide, there are 657 million women aged 45-59 and around half contribute to the labor force during their menopausal years. There is a diversity of experience of menopause in the workplace. It is shaped not only by menopausal symptoms and context but also by the workplace environment. It affects quality of life, engagement, performance, motivation and relations with employers.To provide recommendations for employers, managers, healthcare professionals and women to make the workplace environment more menopause supportive, and to improve women's wellbeing and their ability to remain in work.Literature review and consensus of expert opinion.Workplace health and wellbeing frameworks and policies should incorporate menopausal health as part of the wider context of gender and age equality and reproductive and post-reproductive health. Workplaces should create an open, inclusive and supportive culture regarding menopause, involving, if available, occupational health professionals and human resource managers working together. Women should not be discriminated against, marginalized or dismissed because of menopausal symptoms. Health and allied health professionals should recognize that, for some women, menopausal symptoms can adversely affect the ability to work, which can lead to reduction of working hours, underemployment or unemployment, and consequently financial insecurity in later life.

The prevalence of urinary incontinence and of other lower urinary tract symptoms increases after the menopause and affects between 38 % and 55 % of women aged over 60 years. While urinary incontinence has a profound impact on quality of life, few affected women seek care.The aim of this clinical guide is to provide an evidence-based approach to the management of urinary incontinence in postmenopausal women.Literature review and consensus of expert opinion.Healthcare professionals should consider urinary incontinence a clinical priority and develop appropriate diagnostic skills. They should be able to identify and manage any relevant modifiable factors that could alleviate the condition. A wide range of treatment options is available. First-line management includes lifestyle and behavioral modification, pelvic floor exercises and bladder training. Estrogens and other pharmacological interventions are helpful in the treatment of urgency incontinence that does not respond to conservative measures. Third-line therapies (e.g. sacral neuromodulation, intravesical onabotulinum toxin-A injections and posterior tibial nerve stimulation) are useful in selected patients with refractory urge incontinence. Surgery should be considered in postmenopausal women with stress incontinence. Midurethral slings, including retropubic and transobturator approaches, are safe and effective and should be offered.

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.

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.

Endothelin-1 (ET-1) and its receptors (ET(A)R and ET(B)R), referred to as the endothelin (ET) axis, are overexpressed in breast carcinomas, and influence tumorigenesis and tumor progression by various mechanisms, including angiogenesis. The objective of the study was to clarify if expression of the ET axis participates in angiogenesis of breast carcinomaWe analyzed expression of ET-1, ET(A)R, ET(B)R, and vascular endothelial growth factor (VEGF) immunohistochemically in 600 tissue array specimens from 200 paraffin-embedded breast carcinomas performing tissue microarray technology. Microvessel density (MVD) was determined by counting microvessels (identified by factor VIII) in each core specimen.Moderate or strong immunostaining was observed for ET-1 in 25.4%, for ET(A)R in 43.7%, and for ET(B)R in 22.2% of breast carcinomas. Of all cases, 44.7% showed significant expression of VEGF. MVD varied between different tumor specimens (range, 0-80; median, 17). We observed a statistically significant correlation between MVD and ET expression status with higher MVD in ET-positive tumors. Moreover, expression of VEGF was found more frequently in tumors with overexpression of the ET axis (each P < 0.001). Staining of VEGF was correlated positively with MVD CONCLUSIONS: These results indicate that increased ET-1, ET(A)R, and ET(B)R expression is associated with increased VEGF expression and higher vascularity of breast carcinomas and, thus, could be involved in the regulation of angiogenesis in breast cancer. Our findings provide evidence that the expression pattern of the ET-axis and in particular of ET(A)R may have clinical relevance in future antiangiogenic targeted therapies for breast cancer.

A total of 150 infertile couples underwent chromosome analysis and genetic counselling before intracytoplasmic sperm injection (ICSI). Chromosomal abnormalities, including low-level sex chromosome mosaicism, were detected in 12% of the men and an unexpectedly high 6% of the women. Chromosomal abnormalities included gonosomal mosaicism in 13 cases, Robertsonian translocations in four males, autosomal reciprocal translocations in five cases, reciprocal translocation involving a sex chromosome in one case, inversions in three cases and a marker chromosome in one male. Chromosomal variants found in 11 women and 13 men were not included in the above percentages. Couples with a chromosomal aberration in one partner received a second counselling. The different aspects of genetic counselling in these couples are discussed. In conclusion, we recommend genetic counselling and chromosomal analysis of men and women prior to ICSI therapy.

Clinical EndocrinologyVolume 56, Issue 6 p. 677-687 Clinical use of GnRH analogues L. A. Kiesel, Corresponding Author L. A. Kiesel Departments of Obstetrics and Gynaecology, University of Muenster, Germany and Prof. L. Kiesel, Department of Gynecology and Obstetrics, University of Muenster, Albert-Schweitzer-Str. 33, D-48129 Muenster, Germany. Tel.: + 49 2 51 83 4 82 02; Fax: + 49 2 51 83 4 82 67; E-mail: l.kiesel@uni-muenster.deSearch for more papers by this authorA. Rody, A. Rody Departments of Obstetrics and Gynaecology, University of Muenster, Germany andSearch for more papers by this authorR. R. Greb, R. R. Greb Departments of Obstetrics and Gynaecology, University of Muenster, Germany andSearch for more papers by this authorA. Szilágyi, A. Szilágyi University of Pécs, HungarySearch for more papers by this author L. A. Kiesel, Corresponding Author L. A. Kiesel Departments of Obstetrics and Gynaecology, University of Muenster, Germany and Prof. L. Kiesel, Department of Gynecology and Obstetrics, University of Muenster, Albert-Schweitzer-Str. 33, D-48129 Muenster, Germany. Tel.: + 49 2 51 83 4 82 02; Fax: + 49 2 51 83 4 82 67; E-mail: l.kiesel@uni-muenster.deSearch for more papers by this authorA. Rody, A. Rody Departments of Obstetrics and Gynaecology, University of Muenster, Germany andSearch for more papers by this authorR. R. Greb, R. R. Greb Departments of Obstetrics and Gynaecology, University of Muenster, Germany andSearch for more papers by this authorA. Szilágyi, A. Szilágyi University of Pécs, HungarySearch for more papers by this author First published: 19 June 2002 https://doi.org/10.1046/j.1365-2265.2002.01291.xCitations: 58Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume56, Issue6June 2002Pages 677-687 RelatedInformation

In recent years, preoperative volume reduction of locally advanced breast cancers, resulting in higher rates of breast-conserving surgery (BCS), has become increasingly important also in postmenopausal women. Clinical interest has come to center on the third-generation nonsteroidal aromatase inhibitors (AIs), including letrozole, for such neoadjuvant endocrine treatment. This usually lasts 3-4 months and has been extended to up to 12 months, but optimal treatment duration has not been fully established.This study was designed as a multicenter, open-label, single-arm, exploratory phase IIb/III clinical trial of letrozole 2.5 mg, one tablet daily, for 4-8 months. The primary objective was to investigate the effect of neoadjuvant treatment duration on tumor regression and BCS eligibility to identify optimal treatment duration. Tumor regression (by clinical examination, mammography, and ultrasound), shift towards BCS eligibility, and safety assessments were the main outcome measures. Standard parametric and nonparametric descriptive statistics were performed.Letrozole treatment was received by 32 of the enrolled 33 postmenopausal women (median (range): 67.0 (56-85) years) with unilateral, initially BCS-ineligible primary breast cancer (clinical stage > or = T2, N0, M0). Letrozole treatment duration in the modified intent-to-treat (ITT; required 4 months' letrozole treatment) analysis population (29 patients) was 4 months in 14 patients and > 4 months in 15 patients. The respective per-protocol (PP) subgroup sizes were 14 and 11. The majority of partial or complete responses were observed at 4 months, though some beneficial responses occurred during prolonged letrozole treatment. Compared with baseline, median tumor size in the ITT population was reduced by 62.5% at Month 4 and by 70.0% at final study visit (Individual End). Similarly, in the PP population, respective reductions were 64.0% and 67.0%. Whereas initially all patients were mastectomy candidates, letrozole treatment enabled BCS (lumpectomy) in 22 ITT (75.9%) and 18 PP (72.0%) patients.Over half of patients become BCS-eligible within 4 months of preoperative letrozole treatment. While prolonged treatment for up to 8 months can result in further tumor volume reduction in some patients, there is no clear optimum for treatment duration. Letrozole has a favorable overall safety and tolerability profile.ClinicalTrials.gov identifier NCT00535418.

The mechanism of action of gonadotropin-releasing hormone (GnRH) upon pituitary luteinizing hormone (LH) secretion has not yet been elucidated, but recent evidence has suggested that arachidonic acid or its metabolites are involved in GnRH action. In cultured rat pituitary cells, arachidonic acid and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) elicited concentration-dependent release of LH with EC50 of about 12μM. Other lipoxygenase derivatives including 11-, 12- and 15-HETE, had no consistent effect on LH release, and leukotrienes (B4 and C4) exerted only minor stimulatory actions on LH release. The lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and 3-amino-1-(3-trifluoromethyl phenyl)-2-pyrazoline hydrochloride (BW 755C) caused dose-dependent inhibition of GnRH-induced LH release, with IC50 values of 5, 8.5, and 175μM, respectively. In contrast, the cyclooxygenase inhibitor, indomethacin, had a biphasic action on GnRH-stimulated LH release, with potentiation of GnRH action at low doses (up to 25 μM) and no effect at higher concentrations. These findings are consistent with the potential role of a 5-lipoxygenase product of arachidonic acid in the mechanism of action of GnRH on pituitary gonadotropin release.

Syndecan-1 (Sdc1) plays a major role in wound healing and modulates inflammatory responses. Sdc1 expression is reduced in lesions of patients with ulcerative colitis. The aim of this study was to investigate the role of Sdc1 in murine dextran sodium sulfate (DSS)-induced colitis. DSS colitis was induced in Sdc1-deficient (knockout (KO)) and wild-type mice by oral administration of 3% DSS. KO mice exhibited a significantly increased lethality as compared with wild-type controls (61 versus 5%, P < 0.05). Impaired mucosal healing and prolonged recruitment of inflammatory cells in KO mice were accompanied by significant up-regulation of tumor necrosis factor-α, CC chemokine ligand 3/macrophage inflammatory protein-1α, and vascular cell adhesion molecule-1, as determined by histological correlation between 0 and 15 days after colitis induction, TaqMan low-density array analysis, and quantitative real-time PCR. Treatment from days 7 through 14 with enoxaparin, a functional analogue of the Sdc1 heparan sulfate chains, significantly reduced lethality of KO mice due to DSS-induced colitis, which was correlated with improved mucosal healing. In vitro, Sdc1-deficient polymorphonuclear cells displayed increased adhesion to endothelial cells and intercellular adhesion molecule-1, and enoxaparin reverted adhesion to wild-type levels. Small interfering RNA-mediated knockdown of Sdc1 expression resulted in reduced basic fibroblast growth factor-mediated mitogen-activated protein kinase signaling and reduced Caco-2 cell proliferation. We conclude that Sdc1 has a protective effect during experimental colitis. The modification of missing Sdc1 function by heparin analogues may emerge as a promising anti-inflammatory approach. Syndecan-1 (Sdc1) plays a major role in wound healing and modulates inflammatory responses. Sdc1 expression is reduced in lesions of patients with ulcerative colitis. The aim of this study was to investigate the role of Sdc1 in murine dextran sodium sulfate (DSS)-induced colitis. DSS colitis was induced in Sdc1-deficient (knockout (KO)) and wild-type mice by oral administration of 3% DSS. KO mice exhibited a significantly increased lethality as compared with wild-type controls (61 versus 5%, P < 0.05). Impaired mucosal healing and prolonged recruitment of inflammatory cells in KO mice were accompanied by significant up-regulation of tumor necrosis factor-α, CC chemokine ligand 3/macrophage inflammatory protein-1α, and vascular cell adhesion molecule-1, as determined by histological correlation between 0 and 15 days after colitis induction, TaqMan low-density array analysis, and quantitative real-time PCR. Treatment from days 7 through 14 with enoxaparin, a functional analogue of the Sdc1 heparan sulfate chains, significantly reduced lethality of KO mice due to DSS-induced colitis, which was correlated with improved mucosal healing. In vitro, Sdc1-deficient polymorphonuclear cells displayed increased adhesion to endothelial cells and intercellular adhesion molecule-1, and enoxaparin reverted adhesion to wild-type levels. Small interfering RNA-mediated knockdown of Sdc1 expression resulted in reduced basic fibroblast growth factor-mediated mitogen-activated protein kinase signaling and reduced Caco-2 cell proliferation. We conclude that Sdc1 has a protective effect during experimental colitis. The modification of missing Sdc1 function by heparin analogues may emerge as a promising anti-inflammatory approach. Syndecan-1 (Sdc1) is the most important representative of the heparan sulfate proteoglycans (HSPGs) covering epithelial cell surfaces.1Bernfield M Götte M Park PW Reizes O Fitzgerald ML Lincecum J Zako M Functions of cell surface heparan sulfate proteoglycans.Annu Rev Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2224) Google Scholar It serves multiple biological roles, such as cell-matrix interactions, modulation of inflammatory responses, tumorigenesis, and wound healing.2Götte M Syndecans in inflammation.FASEB J. 2003; 6: 575-5913Crossref Scopus (268) Google Scholar, 3Yip GW Smollich M Götte M Therapeutic value of glycosaminoglycans in cancer.Mol Cancer Ther. 2006; 5: 2139-2148Crossref PubMed Scopus (199) Google Scholar, 4Bishop JR Schuksz M Esko JD Heparan sulfate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1147) Google Scholar The highly conserved cytoplasmic domains of Sdc1 interact with scaffolding proteins and participate in integrin-mediated signaling events, thus providing a physical and functional link to the cytoskeleton. In addition, most of the extracellular-binding interactions are mediated by the heparan sulfate chains, which are structurally and functionally related to heparin, an extensively sulfated and epimerized derivative of heparan sulfate.1Bernfield M Götte M Park PW Reizes O Fitzgerald ML Lincecum J Zako M Functions of cell surface heparan sulfate proteoglycans.Annu Rev Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2224) Google Scholar Sdc1 serves as a coreceptor for several tyrosine kinase receptors. For example, it increases the activity of the complex of basic fibroblast growth factor (bFGF) and the FGF receptor and, therefore, contributes to improved wound healing via stimulation of keratinocyte proliferation.1Bernfield M Götte M Park PW Reizes O Fitzgerald ML Lincecum J Zako M Functions of cell surface heparan sulfate proteoglycans.Annu Rev Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2224) Google Scholar, 5Stepp MA Gibson HE Gala PH Iglesia DD Pajoohesh-Ganji A Pal-Ghosh S Brown M Aquino C Schwartz AM Goldberger O Hinkes MT Bernfield M Defects in keratinocyte activation during wound healing in syndecan-1 deficient mouse.J Cell Sci. 2002; 115: 4517-4531Crossref PubMed Scopus (181) Google Scholar A role for Sdc1 in wound repair in vivo has been demonstrated in Sdc1-deficient (Sdc1-knockout (KO)) mice, which show delayed skin and corneal wound healing5Stepp MA Gibson HE Gala PH Iglesia DD Pajoohesh-Ganji A Pal-Ghosh S Brown M Aquino C Schwartz AM Goldberger O Hinkes MT Bernfield M Defects in keratinocyte activation during wound healing in syndecan-1 deficient mouse.J Cell Sci. 2002; 115: 4517-4531Crossref PubMed Scopus (181) Google Scholar and functionally adverse repair following experimental myocardial infarction due to dysregulation of chemokine expression and matrix metalloproteinase-mediated tissue remodeling.6Vanhoutte D Schellings MW Götte M Swinnen M Herias V Wild MK Vestweber D Chorianopoulos E Cortés V Rigotti A Stepp MA Van de Werf F Carmeliet P Pinto YM Heymans S Increased expression of syndecan-1 protects against cardiac dilatation and dysfunction after myocardial infarction.Circulation. 2007; 115: 475-482Crossref PubMed Scopus (96) Google Scholar Sdc1 forms chemotactic gradients due to binding of chemokines on heparan sulfate chains of the molecule. Therefore, Sdc1 is able to act as coreceptor for chemokine signaling.7Li Q Park PW Wilson CL Parks WC Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury.Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 8Götte M Syndecans as modulators of chemokine function.in: Linkes WP Progress in Chemokine Research. Nova Science Publishers, Hauppauge, NY2007: 85Google Scholar In addition, endothelial leukocyte recruitment and extravasation is modulated by Sdc1, possibly via interference with heparin-binding adhesion molecule function.9Götte M Bernfield M Joussen AM Increased leukocyte-endothelial interactions in syndecan-1 deficient mice involve heparan-sulfate dependent and independent steps.Curr Eye Res. 2005; 6: 417-422Crossref Scopus (27) Google Scholar, 10Götte M Joussen AM Klein C Andre P Wagner DD Hinkes MT Kirchhof B Adamis AP Bernfield M Role of syndecan-1 in leukocyte-endothelial interactions in the ocular vasculature.Invest Ophthalmol Vis Sci. 2002; 43: 1135-1141PubMed Google Scholar, 11Parish CR Heparan sulfate and inflammation.Nat Immunol. 2005; 6: 861-862Crossref PubMed Scopus (40) Google Scholar, 12Kharabi Masouleh B Ten Dam GB Wild MK Seelige R van der Vlag J Rops AL Echtermeyer FG Vestweber D van Kuppevelt TH Kiesel L Götte M Role of the heparan sulfate proteoglycan syndecan-1 (CD138) in delayed-type hypersensitivity.J Immunol. 2009; 182: 4985-4993Crossref PubMed Scopus (45) Google Scholar Day et al13Day R Ilyas M Daszak P Talbot I Forbes A Expression of syndecan-1 in inflammatory bowel disease and a possible mechanism of heparin therapy.Dig Dis Sci. 1999; 44: 2508-2515Crossref PubMed Scopus (48) Google Scholar described in 1999 a reduced expression of Sdc1 in patients with ulcerative colitis, which was linked to disrupted healing of colonic ulcers. In addition, this group demonstrated the benefit of the Sdc1 ectodomain for the FGF-induced proliferation of intestinal epithelial cell lines in vitro. The function of Sdc1 could be restored with heparin, representing a highly sulfated and epimerized form of heparan sulfate, the major functional constituent of the Sdc1 ectodomain. Heparin sees widespread use as anticoagulant drug based on its antithrombin III-activating properties. Enoxaparin, is a low molecular weight heparin with similar features in vitro and in vivo like heparin; however, it exhibits a more favorable pharmacological side effect profile. Both low molecular weight heparins (enoxaparin) as well as heparin were recently found to possess anti-inflammatory properties.14Pellequer Y Meissner Y Ubrich N Lamprecht A Epithelial heparin delivery via microspheres mitigates experimental colitis in mice.J Pharmacol Exp Ther. 2007; 321: 726-733Crossref PubMed Scopus (36) Google Scholar The hypothesis of Sdc1 being involved in the pathogenesis of ulcerative colitis is underlined by multiple clinical observations of patients who have been treated with heparins for different reasons.15Day R Forbes A Heparin, cell adhesion, and pathogenesis of inflammatory bowel disease.Lancet. 1999; 354: 62-6514Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar In a number of cases, this treatment has lead to an improved course of disease. A limited number of uncontrolled clinical trials with heparins in the treatment of low to medium active ulcerative colitis showed a variable outcome,16Evans RC Wong VS Morris AI Rhodes JM Treatment of corticosteroid-resistant ulcerative colitis with heparin—a report of 16 cases.Aliment Pharm Ther. 1997; 1: 1037-1040Crossref Scopus (128) Google Scholar, 17Gaffney P Caffney Doyle A Hogan J Hayes D Annis P Paradoxical response to heparin in 10 patients with ulcerative colits.Am J Gastroenterol. 1995; 90: 220-223PubMed Google Scholar, 18Malhotra S Kondal A Shafiq N Sidhu S Bhasin DK Pandhi P A comparison of observational and controlled trials of heparin in ulcerative colitis.Int J Clin Pharmacol Ther. 2004; 42: 690-694Crossref PubMed Scopus (11) Google Scholar which may be explained by variations in treatment regimes that may have failed to include the optimal dose, class of heparin, and mode of delivery. For example, most studies have involved either i.v. or s.c. delivery of heparin, whereas a more appropriate mode of delivery for stimulating mucosal healing might be the topical application or microsphere-mediated delivery of heparin.14Pellequer Y Meissner Y Ubrich N Lamprecht A Epithelial heparin delivery via microspheres mitigates experimental colitis in mice.J Pharmacol Exp Ther. 2007; 321: 726-733Crossref PubMed Scopus (36) Google Scholar, 15Day R Forbes A Heparin, cell adhesion, and pathogenesis of inflammatory bowel disease.Lancet. 1999; 354: 62-6514Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Furthermore, the outcome of heparin therapy may depend on the degree to which Sdc1 expression is reduced in inflammatory bowel disease (IBD) patients.19Bode L Eklund EA Murch S Freeze HH Heparan sulfate depletion amplifies TNF-α-induced protein leakage in an in vitro model of protein-losing enteropathy.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G1015-G1023Crossref PubMed Scopus (43) Google Scholar Moreover, the expression of Sdc1 and the proinflammatory cytokine tumor necrosis factor-α (TNF-α) are inversely correlated in the colonic mucosa of patients with Crohn's disease,20Principi M Day R Marangi S Burattini O De Francesco V Ingrosso M Pisani A Panella C Forbes A Di Leo A Francavilla A Ierardi E Differential immunohistochemical expression of syndecan-1 and tumor necrosis factor α in colonic mucosa of patients with Crohn's disease.Immunopharmacol Immunotoxicol. 2006; 28: 185-195PubMed Google Scholar and a reduction of Sdc1 expression has been shown to result in increased TNF-α signaling in an in vitro model of protein-losing enteropathy,19Bode L Eklund EA Murch S Freeze HH Heparan sulfate depletion amplifies TNF-α-induced protein leakage in an in vitro model of protein-losing enteropathy.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G1015-G1023Crossref PubMed Scopus (43) Google Scholar, 21Bode L Salvestrini C Park PW Li JP Esko JD Yamaguchi Y Murch S Freeze HH Heparan sulfate plays a central role in a dynamic in vitro model of protein-losing enteropathy.J Biol Chem. 2006; 281: 7809-7815Crossref PubMed Scopus (54) Google Scholar further suggesting a regulatory role for Sdc1 in proinflammatory cytokine signaling. In this study, our goal was to characterize the impact of a Sdc1 deficiency on the dextran sodium sulfate (DSS)-induced colitis of the mouse. Furthermore, the efficacy of low molecular weight heparin to restore altered wound healing was studied in vivo. In addition, in vitro trials were performed to study the role of Sdc1 deficiency in the adhesion and transmigration of leukocytes under inflammatory conditions. Sdc1-deficient (Sdc1 KO) mice on a C57BL/6 background5Stepp MA Gibson HE Gala PH Iglesia DD Pajoohesh-Ganji A Pal-Ghosh S Brown M Aquino C Schwartz AM Goldberger O Hinkes MT Bernfield M Defects in keratinocyte activation during wound healing in syndecan-1 deficient mouse.J Cell Sci. 2002; 115: 4517-4531Crossref PubMed Scopus (181) Google Scholar, 12Kharabi Masouleh B Ten Dam GB Wild MK Seelige R van der Vlag J Rops AL Echtermeyer FG Vestweber D van Kuppevelt TH Kiesel L Götte M Role of the heparan sulfate proteoglycan syndecan-1 (CD138) in delayed-type hypersensitivity.J Immunol. 2009; 182: 4985-4993Crossref PubMed Scopus (45) Google Scholar and control wild-type mice were obtained from the clinical research laboratory of the Department of Gynecology, University of Muenster. Mice were bred, housed, and handled according to the guidelines of the local animal ethics committee (number A101/2005). Twelve-week-old male mice were placed in the Central Animal Facility of the University Hospital of Muenster with a 12-hour day/night light cycle and standard chow and water ad libitum. After 3 weeks of adaptation, the trials were performed as follows: starting on day 1 and continued to day 6 an oral administration of 3% DSS in drinking water was administered. On day 7, the water was switched to normal drinking water. Of 92 mice used in this study, 10 male wild-type mice and 10 Sdc1 KO mice were used to study the course of disease. The course of disease was monitored by daily weight measurement and a daily assessment of blood in stool with a commercially available stool test (Hemoccult; Beckman Coulter, Fullerton, CA). Thirty-six wild-type mice and 36 Sdc1 KO mice were used for histological evaluation and for real-time PCR analysis of colon tissue divided in a treatment group of 18 mice and a control group of another 18 mice. The treatment group received an i.p. injection of enoxaparin (500 IU/kg/body weight) from days 7 to 14 (control group: 0.9% NaCl) once per day. Three animals out of each group were sacrificed for further histological examination on days 0, 3, 6, 9, 12, and 15. After euthanasia of animals, the proximal, medial, and distal colon was resected after laparotomy. The colon was flushed with PBS and covered with optimal cutting temperature Tissue-Tek (Sakura). The tissue was snap frozen in liquid nitrogen and stored at −80°C until use. Seven-micrometer frozen sections were cut, and standard H&E staining was performed as described previously.22Anthoni C Laukoetter MG Rijcken E Vowinkel T Mennigen R Müller S Senninger N Russell J Jauch J Bergmann J Granger DN Krieglstein CF Mechanisms underlying the anti-inflammatory actions of boswellic acid derivates in experimental colitis.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1131-G1137Crossref PubMed Scopus (90) Google Scholar Severity of the colitis was assessed by an established scoring system as published before.23Hausmann M Obermeier F Paper DH Balan K Dunger N Menzel K Falk W Schoelmerich J Herfarth H Rogler G In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulfate sodium-induced colitis.Clin Exp Immunology. 2007; 148: 373-381Crossref PubMed Scopus (110) Google Scholar For immunohistochemical analysis, the following primary antibodies were used: Sdc1 (rat anti-mouse Sdc1 mAb clone 281-2, diluted 1/100 in PBS with 1% BSA; BD Pharmingen, Heidelberg, Germany), Sdc2 (rabbit-anti-mouse, diluted 1/50; Santa Cruz Biotechnology, Santa Cruz, CA), Sdc3 (rat-anti-mouse mAb clone 312607, diluted 1/50; R&D Systems, Wiesbaden, Germany), Sdc4 (rat-anti-mouse clone KY/8.2, diluted 1/50; BD Pharmingen), F4/80 (rat-anti-mouse, clone BM8; eBioscience, San Diego, CA), CD4, CD8, and B220 (rat anti-mouse, clone C3T4 (CD4), clone 53-6.7 (CD8), and clone RA3-6B2 (B220), diluted 1/100; BD Pharmingen). M cells were stained with the UEA1 lectin method (Sigma-Aldrich, Deisenheim, Germany) as described elsewhere.23Hausmann M Obermeier F Paper DH Balan K Dunger N Menzel K Falk W Schoelmerich J Herfarth H Rogler G In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulfate sodium-induced colitis.Clin Exp Immunology. 2007; 148: 373-381Crossref PubMed Scopus (110) Google Scholar Briefly, the sections were blocked with 10% goat serum or 1% BSA (Dako, Glostrup, Denmark) in PBS for 30 minutes. Incubation with the primary antibody was performed in a humid chamber overnight at 4°C. After three washes with PBS (5 minutes), the secondary antibody (dilution 1/1000 in 10% goat serum/PBS) was incubated for 1 hour at room temperature. For immunofluorescence microscopy, Alexa Fluor-labeled secondary antibodies were used (Invitrogen, Karlsruhe, Germany), while for conventional immunohistochemistry, the Vectastain ABC system (Vector Laboratories, Burlingame, CA) or the Dako EnVision system (Dako) were used, depending on the primary antibody used. For immunofluorescence microscopy, sections were washed again in PBS for 5 minutes, air-dried, and treated with prolong antifade kit (Invitrogen). For conventional immunohistochemistry, 3-amino-9- ethylcarbazole substrate (Dako) was used for color development, followed by Mayer's Hemalum counterstaining (Merck, Darmstadt, Germany). Pictures were taken with a Leica or Zeiss Axioscope microscope, and digital cameras directly connected to a PC system. Images were processed with Adobe Photoshop version 7.0 and Zeiss Axiovision software. To study the composition of intestinal lymphoid subpopulations, flow cytometry was performed.24Lügering A Floer M Westphal S Maaser C Spahn TW Schmidt MA Domschke W Williams IR Kucharzik T Absence of CCR6 inhibits CD4+ regulatory T cell development and M cell formation in inside Peyer's patches.Am J Pathol. 2005; 166: 1647-1654Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Briefly, Peyer's patches (PPs) and lamina propria lymphocytes from the colon were extracted and analyzed by flow cytometry. All PPs were counted and resected from the intestine. PPs were homogenized mechanically with a Falcon cell mesh (pore size, 70 μm; BD Pharmingen) and mixed with ice-cold RPMI 1640 medium and 10% fetal calf serum. After centrifugation at 10,000 × g for 5 minutes, the supernatant was discarded, and the pellet was washed with ice-cold PBS. This washing/centrifugation step was performed 3 times. The pellet was resuspended in ice-cold PBS and added to Ficoll (Genaxxon Bioscience, Biberach, Germany). After centrifugation at 5000 × g for 15 minutes, the layer containing the leukocytes was retrieved and placed into a fluorescence-activated cell sorting tube. After a washing step with PBS and centrifugation, the pellet was incubated with an antibody for surface markers CD4 (direct fluorescein isothiocyanate-conjugated mAb, clone C3T4; BD Pharmingen), CD8 (direct phycoerythrin-conjugated mAb, clone 53-6.7; BD Pharmingen), and B220 (direct allophycocyanin-conjugated mAb, clone RA3-6B2; BD Pharmingen). Furthermore, in lamina propria lymphocyte populations, the markers CD3 (clone 145-2C11), CD11c (clone HC3), CD25 (clone 7D4), CD127 (clone A7R34), and Gr-1 (clone RB6-8C5) (all antibodies purchased from BD Pharmingen) were examined. Fluorescence-activated cell sorting analysis was performed with a FACSCalibur flow cytometer from BD Biosciences (Heidelberg, Germany). The human colon carcinoma cell line Caco-2 (German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany) was cultured in minimal essential medium (Invitrogen) containing 10% fetal calf serum, 1% penicillin/streptomycin and 1 mm of l-glutamine. Small interfering RNA (siRNA)-mediated knockdown of Sdc1 expression was performed exactly as previously described,25Nikolova V Koo CY Ibrahim SA Wang Z Spillmann D Dreier R Kelsch R Fischgräbe J Smollich M Rossi LH Sibrowski W Wülfing P Kiesel L Yip GW Götte M Differential roles for membrane-bound and soluble syndecan-1 (CD138) in breast cancer progression.Carcinogenesis. 2009; 30: 397-407Crossref PubMed Scopus (136) Google Scholar using siRNA numbers 12527 and 12432 (Ambion, Cambridgeshire, UK) targeting the coding region of Sdc1 and a negative control siRNA (number 301698; Qiagen, Hilden, Germany) at 40 nmol/L and Dharmafect reagent (Dharmacon, Lafayette, CO). Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assay essentially as previously described,26Seidler DG Faiyaz-Ul-Haque M Hansen U Yip GW Zaidi SH Teebi AS Kiesel L Götte M Defective glycosylation of decorin and biglycan, altered collagen structure, and abnormal phenotype of the skin fibroblasts of an Ehlers-Danlos syndrome patient carrying the novel Arg270Cys substitution in galactosyltransferase I (β4GalT-7).J Mol Med. 2006; 84: 583-594Crossref PubMed Scopus (89) Google Scholar using 5000 Caco-2 cells/well plated 2 days after Sdc1 silencing for an incubation period of 3 days. bFGF-mediated mitogen-activated protein kinase (MAPK) activation was studied 72 hours after Sdc1 knockdown. Silenced serum-starved Caco-2 cells were stimulated with 20 nmol/L bFGF (R&D Systems, Wiesbaden, Germany) for 0, 10, or 20 minutes, respectively, and cell extracts were subjected to SDS-PAGE and Western blotting for phosphorylated and total p44/42 MAPK using rabbit polyclonal antibodies (Cell Signaling Technology, Beverly, MA) exactly as described previously.25Nikolova V Koo CY Ibrahim SA Wang Z Spillmann D Dreier R Kelsch R Fischgräbe J Smollich M Rossi LH Sibrowski W Wülfing P Kiesel L Yip GW Götte M Differential roles for membrane-bound and soluble syndecan-1 (CD138) in breast cancer progression.Carcinogenesis. 2009; 30: 397-407Crossref PubMed Scopus (136) Google Scholar All experiments were performed at least three times. Polymorphonuclear (PMN) cells were purified from the bone marrow of Sdc1-deficient and wild-type mice by Histopaque (Sigma-Aldrich) gradient centrifugation as previously described,11Parish CR Heparan sulfate and inflammation.Nat Immunol. 2005; 6: 861-862Crossref PubMed Scopus (40) Google Scholar and leukocyte adhesion to the murine bEnd.3 endothelial cell line, derived from viral oncogene-immortalized brain endothelial cells27Montesano R Pepper MS Möhle-Steinlein U Risau W Wagner EF Orci L Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.Cell. 1990; 62: 435-445Abstract Full Text PDF PubMed Scopus (362) Google Scholar was measured under static conditions. Briefly, PMN cells were fluorescently labeled with 2,7 -bis-(2 carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester (Invitrogen), washed, and incubated in sextuplets (2 × 106 cells/ml, 50 μl/well) with confluent bEnd.3 monolayers in 96-well plates (10 minutes, 37°C). For inhibition experiments, PMN cells were preincubated for 30 minutes with 10 or 25 μg/ml enoxaparin, followed by incubation in the presence of the drug as described above. After three washes with PBS, adhering cells were lysed, and the fluorescence signal was quantified in a Spectramax fluorimeter (excitation, 485 nm; emission, 535 nm). Statistical analysis was performed using Student's t-test. A value of P < 0.05 was considered statistically significant. Adhesion of Sdc1-deficient and wild-type mouse PMN cells to recombinant Intercellular adhesion molecule-1 (ICAM-1) was performed essentially as described.28Varga G Balkow S Wild MK Stadtbaeumer A Krummen M Rothoeft T Higuchi T Beissert S Wethmar K Scharffetter-Kochanek K Vestweber D Grabbe S Active MAC-1 (CD11b/CD18) on DCs inhibits full T cell activation.Blood. 2007; 109: 661-669Crossref PubMed Scopus (89) Google Scholar Briefly, 10 μg/ml recombinant murine ICAM-1 protein in PBS/1% fetal calf serum was coated onto 96-well flat-bottom plates (Maxisorp; Nunc, Wiesbaden, Germany). Murine PMN cells, prepared as described above, were added and incubated at a concentration of 3 × 105 cells/well for 45 minutes at 4°C to allow for cell-ICAM-1 interactions. For inhibition experiments, PMN cells were preincubated for 30 minutes with 10 or 25 μg/ml enoxaparin, followed by incubation in the presence of the drug as described above. Plates were washed four times with PBS, and adherent cells were fixed using 1% paraformaldehyde (w/v) in PBS. Adhering cells were observed at 32-fold magnification using a Zeiss Axioskop microscope (Zeiss, Göttingen, Germany) equipped with a charge-coupled device camera. Cell numbers were quantified and expressed as cells/mm2 with the assistance of Zeiss Axiovision imaging software. Statistical analysis was performed using Student's t-test. A value of P < 0.05 was considered statistically significant. Granulocytes were incubated with 10 μg/ml E- and P-selectin-Fc proteins consisting of the extracellular parts of the murine selectins and the Fc portion of human IgG essentially as described previously.29Hahne M Jäger U Isenmann S Hallmann R Vestweber D Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.J Cell Biol. 1993; 121: 655-664Crossref PubMed Scopus (197) Google Scholar, 30Gotsch U Borges E Bosse R Boggemeyer E Simon M Mossmann H Vestweber D VE-cadherin antibody accelerates neutrophil recruitment in vivo.J Cell Sci. 1997; 110: 583-588Crossref PubMed Google Scholar Human IgG1 (Sigma-Aldrich) served as negative control. Before protein incubation, unspecific binding was blocked by incubation with 10 μg/ml Fc block 2.4G2 (BD Pharmingen). Phycoerythrin-conjugated donkey-anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) served to visualize binding using a FACSCanto flow cytometer and FACSDiva software (BD Biosciences). Animals were sacrificed, and the medial to distal colon was resected. Colon tissue was immediately placed in RNAlater solution (Ambion, Huntington, UK), followed by preparation of total RNA using the RNAeasy kit (Qiagen). Five hundred nanograms of total RNA was transcribed using the high-capacity cDNA synthesis kit (Applied Biosystems). cDNA corresponding to 0.5 ng of total RNA was used as a template in the PCR consisting of ABI MasterMix (Applied Biosystems, Darmstadt, Germany) and predesigned TaqMan gene expression systems (Applied Biosystems) or the TaqMan Low-Density Mouse Immune Panel (ABI number 4367786), according to the manufacturers instructions. PCR was performed using 7900HT Fast and 7300 Real-Time PCR Systems (Applied Biosystems). For detection of Sdc1 to Sdc4, P-Selectin, TNF-α, vascular cell adhesion molecule-1 (VCAM-1), ICAM-1, CC chemokine ligand (CCL)3/macrophage inflammatory protein-1α (MIP-1α), CCL2/ monocyte chemoattractant protein-1, interleukin-6 mRNA, primers Mm00448918_m1 (Sdc1 exon 2–3), Mm00484718_m1 (Sdc2 exon 4–5), Mm01179831_m1 (Sdc3 exon 2–3), Mm00488527_m1 (Sdc4 exon 4–5), Mm 00441295_m1 (P-Selectin exon 8–9), Mm00443258_m1 (TNF α exon 1–2), Mm00449197_m1 (VCAM-1 exon 5–6), Mm00516023_m1 (ICAM-1 exon 2–3), Mm00441242_m1 (CCL2 exon 1–2), Mm00441258_m1 (CCL3 exon 1–2), and Mm00446190_m1 (interleukin-6 exon 2–3) were used and normalized to the expression of mammalian 18S rRNA (Hs99999901_s1, all primers from Applied Biosystems). Quantitative real-time PCR was performed using the ABI PRISM 7300 Sequence Detection System (Applied Biosystems) by using the default thermal cycling conditions (10 minutes at 95°C and then 40 cycles of 15 seconds at 95°C plus 1 minute at 60°C). Relative quantification was performed using the comparative cycle threshold method. For low-density array analysis 2 (Sdc1 KO DSS), or 3 (Sdc1 KO control, wild-type control, DSS) biological replicates were used. For quantitative real-time PCR, 5–9 biological replicates were used. For statistical analysis of ΔCt values, Student's t-test was used. A value of P < 0.05 was considered statistically significant. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was used on frozen sections of the colon according to the manufacturers manual (TdT-fragEL; Calbiochem, San Diego, CA).

The discovery of the OPG/RANK/RANKL pathway two decades ago has initiated novel insights into regulation of bone formation. More recently this pathway has been found to be also relevant in osteoclastic-independent mechanisms, mainly in mammary physiology and breast cancer. RANKL/RANK function is essential for epithelial cell proliferation and cellular survival as well as lobulo-alveolar development. The endogenous OPG functions as a soluble decoy receptor, binding the cytokine RANKL to prevent RANKL from activating its receptor RANK. The regulatory function of RANKL is one of the key factors in progesterone-induced proliferation of the breast. Progesterone has a direct action of progesterone on progesterone-receptor (PR) expressing cells but PR-negative cells are affected indirectly through RANKL-induced paracrine actions leading to proliferation of mammary epithelial PR-negative cells. RANK induces epithelial-mesenchymal transition and stemness in human mammary epithelial cells and promotes tumorigenesis and metastasis. Inhibition of the RANK/RANKL pathway using the monoclonal antibody denosumab can neutralize RANKL and inhibiting its interaction with its receptor RANK. Denosumab is currently used to treat osteoporosis and in prevention of skeletal related events in patients suffering from bone metastases due to solid tumors. As preclinical experiments suggest the RANKL/RANK pathway plays an important role in primary breast cancer development. The interference with the RANK/RANKL system could therefore serve as a potential target for prevention and treatment of breast cancer.

Globally, the total number of people with depression exceeds 300 million, and the incidence rate is 70 % greater in women. The perimenopause is considered to be a time of increased risk for the development of depressive symptoms and major depressive episodes.The aim of this position statement is to provide a comprehensive model of care for the management of depressive symptoms in perimenopausal and early menopausal women, including diagnosis, treatment and follow-up. The model integrates the care provided by all those involved in the management of mild or moderate depression in midlife women.Literature review and consensus of expert opinion.Awareness of depressive symptoms, early detection, standardized diagnostic procedures, personalized treatment and a suitable follow-up schedule need to be integrated into healthcare systems worldwide. Recommended treatment comprises antidepressants, psychosocial therapies and lifestyle changes. Alternative and complementary therapies, although widely used, may help with depression, but a stronger evidence base is needed. Although not approved for this indication, menopausal hormone therapy may improve depressive symptoms in peri- but not in postmenopausal women, especially in those with vasomotor symptoms.

Vasomotor symptoms (VMS) (hot flashes and night sweats) affect most women over the menopause transition. Comparing the safety and effectiveness of treatments for vasomotor symptoms is limited by the use of inconsistent outcome measures, and uncertainty as to which outcomes are most important to symptomatic women. To address this, we have developed a Core Outcome Set (COS) for use in clinical trials of treatments for VMS.We systematically reviewed the primary outcomes measured in randomized controlled trials of treatments for VMS. These were refined and entered into a two-round modified Delphi survey completed by clinicians, researchers, and postmenopausal women between November 2019 and March 2020. Outcomes were scored on a nine-point scale from "not important" to "critically important." Two international consensus meetings were held to finalize the COS.Based on the systematic review, 13 separate outcomes were included in the Delphi process. This was completed by 227 participants of whom 58% were postmenopausal women, 34% clinicians, and 8% researchers. Predefined thresholds were applied to categorize importance scores obtained during Round 2 of the Delphi survey. These informed discussions at the consensus meetings which were attended by 56 participants from 28 countries. The final COS includes six outcomes: 1) frequency of VMS, 2) severity of VMS, 3) distress, bother or interference caused by VMS, 4) impact on sleep, 5) satisfaction with treatment, and 6) side-effects of treatment.Implementation of this COS will: better enable research studies to accurately reflect the joint priorities of postmenopausal women, clinicians and researchers, standardize outcome reporting, and facilitate combining and comparing results from different studies, and ultimately improve outcomes for women with bothersome VMS.Video Summary:http://links.lww.com/MENO/A763 .

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.

ABSTRACT Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.

Angiogenesis plays an important role in the development of the ovarian follicle and its subsequent transition into the corpus luteum. Accordingly, follicular fluid is a rich source of mitogenic and angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor secreted by granulosa cells. In the present study, we show that follicular fluid deprived of basic fibroblast growth factor or vascular endothelial growth factor by means of thermal denaturation or antibody neutralization retains its capacity to stimulate endothelial proliferation and angiogenesis. Mass spectrometric analysis of chromatographic fractions stimulating endothelial growth obtained from follicular fluid revealed that the heat-stable mitogenic activity is identical with the subfraction α of high density lipoproteins purified from follicular fluid (FF-HDL). Further investigations demonstrated that sphingosine 1-phosphate (S1P), one of the lysophospholipids associated with HDL, accounts for the capacity of this lipoprotein to stimulate endothelial growth and the formation of new vessels. Activation of mitogen-activated protein kinase (p42/44ERK1/2), protein kinase C, and protein kinase Akt represent signaling pathways utilized by FF-HDL and S1P to induce endothelial proliferation and angiogenesis. We conclude that FF-HDL represents a novel mitogenic and angiogenic factor present in follicular fluid and that S1P is one of the FF-HDL lipid components accounting for this activity. Angiogenesis plays an important role in the development of the ovarian follicle and its subsequent transition into the corpus luteum. Accordingly, follicular fluid is a rich source of mitogenic and angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor secreted by granulosa cells. In the present study, we show that follicular fluid deprived of basic fibroblast growth factor or vascular endothelial growth factor by means of thermal denaturation or antibody neutralization retains its capacity to stimulate endothelial proliferation and angiogenesis. Mass spectrometric analysis of chromatographic fractions stimulating endothelial growth obtained from follicular fluid revealed that the heat-stable mitogenic activity is identical with the subfraction α of high density lipoproteins purified from follicular fluid (FF-HDL). Further investigations demonstrated that sphingosine 1-phosphate (S1P), one of the lysophospholipids associated with HDL, accounts for the capacity of this lipoprotein to stimulate endothelial growth and the formation of new vessels. Activation of mitogen-activated protein kinase (p42/44ERK1/2), protein kinase C, and protein kinase Akt represent signaling pathways utilized by FF-HDL and S1P to induce endothelial proliferation and angiogenesis. We conclude that FF-HDL represents a novel mitogenic and angiogenic factor present in follicular fluid and that S1P is one of the FF-HDL lipid components accounting for this activity. Angiogenesis, the formation of blood vessels from endothelial cells, occurs during embryonic and adult life (1Augustin H.G. Baillieres Best Pract. Res. Clin. Obstet. Gynaecol. 2000; 14: 867-882Crossref PubMed Scopus (49) Google Scholar). One of the few organ systems in healthy adults in which angiogenic processes appear is the female reproductive system. In the cytogenic and hormone-secreting ovaries, angiogenesis occurs on a dynamic basis during the reproductive cycle (2Reynolds L.P. Killilea S.D. Redmer D.A. FASEB J. 1992; 6: 886-892Crossref PubMed Scopus (370) Google Scholar). The transition of the follicle into the corpus luteum and subsequent luteolysis is accompanied by growth and subsequent regression of blood vessels (3Abulafia O. Sherer D.M. Am. J. Obstet. Gynecol. 2000; 182: 240-246Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Massive proliferation and migration of endothelial cells preceding new vessel formation is especially visible during corpus luteum formation after the ovulating gonadotrophin surge (4Meyer G.T. McGeachie J.K. Anat. Rec. 1988; 222: 18-25Crossref PubMed Scopus (33) Google Scholar). These dynamic processes are driven by growth factors and cytokines and are tempered by inhibitors of angiogenesis present in follicular fluid. Several pro- and antiangiogenic molecules involved in endothelial cell proliferation, the principal process underlying ovarian angiogenesis, have been identified in follicular fluid (5Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4866) Google Scholar). These include in the first place basic fibroblast growth factor (bFGF) 3The abbreviations used are: bFGF, basic fibroblast growth factor; apo, apolipoprotein;FF, follicular fluid; FF-HDL, follicular fluid high density lipoprotein; S1P, sphingosine 1-phosphate; HUVEC, human umbilical vein endothelial cell(s); PKC, protein kinase C; VEGF, vascular endothelial growth factor; MALDI, matrix-assisted laser desorption ionization; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. and vascular endothelial growth factor (VEGF) (6Phillips H.S. Hains J. Leung D.W. Ferrara N. Endocrinology. 1990; 127: 965-967Crossref PubMed Scopus (252) Google Scholar, 7Reynolds L.P. Redmer D.A. J. Anim. Sci. 1998; 76: 1671-1681Crossref PubMed Scopus (125) Google Scholar, 8Gospodarowicz D. Cheng J. Lui G.M. Baird A. Esch F. Bohlen P. Endocrinology. 1985; 117: 2383-2391Crossref PubMed Scopus (187) Google Scholar, 9Kamat B.R. Brown L.F. Manseau E.J. Senger D.R. Dvorak H.F. Am. J. Pathol. 1995; 146: 157-165PubMed Google Scholar), as well as angiopoietins 1 and 2 (10Hayashi K.G. Acosta T.J. Tetsuka M. Berisha B. Matsui M. Schams D. Ohtani M. Miyamoto A. Biol. Reprod. 2003; 69: 2078-2084Crossref PubMed Scopus (49) Google Scholar), SPARC (secreted protein acidic and rich in cysteine), and thrombospondin (11Bagavandoss P. Sage E.H. Vernon R.B. J. Histochem. Cytochem. 1998; 46: 1043-1049Crossref PubMed Scopus (35) Google Scholar). The exact contribution of each of these factors to the formation of new vessels within ovarian tissues remains unclear. Our previous investigations of angiogenic constituents of human follicular fluid indicated that a considerable portion of its mitogenic activity cannot be attributed to VEGF or bFGF (12von Otte S. Paletta J.R.J. Burghus B. Rickert-Föhring M. Nordhoff V. Kiesel L. Greb R.R. Hum. Reprod. 2003; 18: 125-126Google Scholar). For instance, VEGF and bFGF added to endothelial cells at concentrations equivalent to those present in follicular fluid were revealed to be several times less potent in inducing endothelial proliferation than follicular fluid itself. Even the combined application of VEGF and bFGF to endothelial cells failed to induce mitogenic response comparable with that triggered by follicular fluid. These data clearly indicate that follicular fluid is a source of angiogenic activity distinct from traditional growth factors. Follicular fluid HDL (FF-HDL) has been identified as a sole lipoprotein present in follicular fluid until the ovulation of the oocyte (13Simpson E.R. Rochelle D.B. Carr B.R. MacDonald P.C. J. Clin. Endocrinol. Metab. 1980; 51: 1469-1471Crossref PubMed Scopus (81) Google Scholar). Unlike serum HDL, FF-HDL particles are cholesterol-poor but contain significantly higher amounts of phospholipids. Apolipoproteins apoA-I and apoA-IV are major protein constituents of FF-HDL. The physiological role fulfilled by FF-HDL remains obscure. It has been proposed that FF-HDL is involved in sperm capacitation (14Therien I. Bousquet D. Manjunath P. Biol. Reprod. 2001; 65: 41-51Crossref PubMed Scopus (57) Google Scholar) or delivery of cholesterol to granulosa cells for progesterone production (15Azhar S. Tsai L. Medicherla S. Chandrasekher Y. Giudice L. Reaven E. J. Clin. Endocrinol. Metab. 1998; 83: 983-991Crossref PubMed Scopus (92) Google Scholar). Here we report that FF-HDL is a potent and endothelial cell-specific mitogen present in human follicular fluid. We further demonstrate that sphingosine 1-phosphate (S1P), the lysosphingolipid identified in FF-HDL, accounts for a significant portion of the mitogenic and angiogenic activity associated with this lipoprotein. Reagents—S1P was purchased from Biomol (Hamburg, Germany). ApoA-I and apoA-IV were obtained from Sigma. The inhibitors PD98059, H7, and LY294002 were purchased from Calbiochem (Schwalbach, Germany). Human Follicular Fluid—Human follicular fluid (FF) was obtained from 10 women undergoing ovarian hyperstimulation with recombinant follicle-stimulating hormone (Gonal F®, Serono, Unterschleissheim, Germany) after pituitary desensitization with nafarelin acetate (Synarela®; Amersham Biosciences) for in vitro fertilization or intracytoplasmic sperm injection. Ovulation induction was performed by administration of 10,000 IU human gonadotrophin (hCG; Choragon®, Ferring, Kiel, Germany) when at least three follicles reached a diameter of at least 18 mm. After 36 h, follicular fluids were aspirated transvaginally under ultrasound guidance. For removal of cell debris, follicular fluids were immediately centrifuged after aspiration for 5 min at 1500 rpm. All samples were frozen at –80 °C for further analysis. For experiments, fluid samples of all aspirated follicles were pooled. Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)—HUVEC were isolated from human umbilical cord veins as described by Jaffe et al. (16Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (6019) Google Scholar) with minor modifications. Briefly, the venous lumen was washed with phosphate-buffered saline to remove coagulated blood, filled with 0.1% collagenase I (Worthington), and incubated for 15 min at 37 °C. The cell suspension was obtained by flushing the lumen with endothelial growth medium (Promocell, Heidelberg, Germany) and centrifuged at 173 × g for 10 min. The supernatant was removed, and the cell pellet was resuspended in endothelial cell medium. Cells were seeded at a density of 24,000 cells/cm2 in cell culture dishes (BD Biosciences, Bedford, MA), cultured at 37 °C in a CO2-enriched atmosphere and in medium containing 2% (w/v) fetal calf serum and gentamicin. Confluent monolayers were passaged twice at a ratio of 1:3 using 0.05% trypsin and 0.02% EDTA. Cells were either frozen at passage 2 or used for experiments in passage 3. Cell identity was confirmed by immunofluorescence microscopy using fluorescein isothiocyanate-labeled antibodies against CD31 (BD Biosciences). Proliferation Assay—Proliferation was assayed in 96-well plates. Cells were seeded at densities of 1 × 104 cells/well in 0.2 ml of endothelial cell medium in the absence or presence of follicular fluid (up to 80 μl/well). After incubation for 48 h at 37 °C, the proliferation was analyzed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) using a commercially available kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Heat Treatment of Follicular Fluid and Immunoneutralization—Heat inactivation was performed by incubation of follicular fluid for 30 min at 90 °C. After cooling, follicular fluid was centrifuged for 60 min at 2500 × g to remove denatured proteins, and the supernatant was retrieved for further processing. For immunoneutralization, anti-human VEGF antibody and anti-bFGF antibody (both from R&D Systems) were added to follicular fluid at an antibody/growth factor molar ratio of 1000:1 and incubated for 1 h at room temperature. The concentrations of bFGF and VEGF in native, heat-treated, and immunoneutralized follicular fluid were determined in duplicates by a quantitative sandwich-enzyme immunoassay technique using a Quantikine human VEGF immunoassay and a Quantikine human bFGF immunoassay (both from R&D Systems). The minimum detection limits of the assays were 5 and 3 pg/ml for VEGF and bFGF, respectively. Purification of the Mitogenic Activity—All steps of the purification were carried out at 4–8 °C. Native follicular fluid (100 ml) was heat-treated and centrifuged as described above. The resulting supernatant (50 ml) was concentrated by ultrafiltration in a pressure chamber (Amicon, Beverly, MA), equipped with an YN30 membrane. The concentrate (5 ml) was applied to a DEAE-Sepharose column (2.6 × 22.0 cm; 116-ml bed volume (Amersham Biosciences) equilibrated with 10 mm Tris/HCl buffer (pH 7.0), with a flow rate of 0.5 bed volume/h. The column was washed with 2.5 bed volumes of buffer, and bound proteins were eluted with a linear NaCl gradient (0–1.0 mol/liter; 600 ml) at a flow rate of 1 ml/min. Fractions exhibiting high protein content were pooled, desalted, and subjected to further analysis. Protein Separation and Identification—Proteins were separated under denaturing conditions in 11.5% (w/w) polyacrylamide gels by the method of Laemmli (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207522) Google Scholar) and stained with Serva Blue R (Serva, Heidelberg, Germany). Bands were excised, and the proteins were tryptically digested in the gel following a slightly modified general procedure, which was previously published (18Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7832) Google Scholar). Peptides generated by proteolysis were extracted, purified using C18 solid phase extraction (ZipZips; Millipore Corp., Bedford, MA), and subjected to MALDI time-of-flight mass spectrometry using TofSpec-2E (Waters/Micromass, Manchester, UK). Data base searches were performed using Mascot software (Matrix Science Ltd., London, UK) screening the NCBI and SwissProt data bases. Lipoprotein Isolation, Characterization, and Modification—HDL (d = 1.125–1.210 g/ml) was isolated from follicular fluid by a discontinuous ultracentrifugation as described by Havel et al. (19Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6498) Google Scholar) and dialyzed overnight against phosphate-buffered saline. The composition of lipoprotein in follicular fluid was investigated using one- or two-dimensional nondenaturing gel electrophoresis. A standard electrophoresis in agarose gel (1% w/v) was performed in the first dimension to separate major lipoprotein classes. The gels were stained with Sudan black and densitometered, or agarose strips were placed on a 2–15% polyacrylamide gradient gel with a 2% stacking gel. The electrophoresis was performed in a 25 mmol/liter Tris, 0.2 mol/liter glycine buffer (pH 8.2) for 3 h at 100 V. After electrophoresis, gels were electrotransferred to nitrocellulose membranes for immunoblotting with anti-apoA-I antibodies. Horseradish peroxidase-conjugated secondary antibodies were used for apolipoprotein visualization in a chemiluminescence-based procedure. To reduce S1P content in HDL isolated from follicular fluid, the treatment with alkalic phosphatase as described by Ruwisch et al. (20Ruwisch L. Schafer-Korting M. Kleuser B. Naunyn. Schmiedeberg's Arch. Pharmacol. 2001; 363: 358-363Crossref PubMed Scopus (65) Google Scholar) was used. Briefly, 50 units of alkaline phosphatase were diluted in 0.45 ml of buffer containing 200 mmol/liter Tris-HCl (pH 4.5) and 75 mmol/liter MgCl2 in glycine (2 mol/liter, pH 9.0) and added to 1.5 ml of HDL. After incubation for 30 min at 37 °C, pH was neutralized with HCl. Control samples were treated identically without the addition of phosphatase. The apoA-I-phospholipid complexes were prepared as described previously (21Nofer J.R. Junker R. Pulawski E. Fobker M. Levkau B. von Eckardstein A. Seedorf U. Assmann G. Walter M. Thromb. Haemost. 2001; 85: 730-735Crossref PubMed Scopus (64) Google Scholar). Western Blotting—HUVEC were lysed in 0.18 mol/liter Tris-HCl, 0.15 mol/liter NaCl, 10% (v/v) Nonidet P-40, 5% (v/v) sodium deoxycholate, 1% (v/v) SDS, 50 mmol/liter, 50 mmol/liter NaF, 1 mmol/liter EGTA, 1 mmol/liter orthovanadate, and the Complete® protease inhibitor mixture. Cell lysates (50 μg/lane) were subjected to SDS-gel polyacrylamide electrophoresis according to Laemmli (15Azhar S. Tsai L. Medicherla S. Chandrasekher Y. Giudice L. Reaven E. J. Clin. Endocrinol. Metab. 1998; 83: 983-991Crossref PubMed Scopus (92) Google Scholar). Thereafter, proteins were transferred to nitrocellulose membranes, which were blocked overnight in Tris-buffered saline containing 5% fat dry milk prior to incubations with antibodies. Loading controls were performed using an antibody against a ubiquitously expressed protein (α-actin). Determination of Sphingosine 1-Phosphate—S1P levels were determined as described previously (20Ruwisch L. Schafer-Korting M. Kleuser B. Naunyn. Schmiedeberg's Arch. Pharmacol. 2001; 363: 358-363Crossref PubMed Scopus (65) Google Scholar). Briefly, HDL was mixed with methanol/HCl (1:1; v/v), and lipids were extracted by the addition of 1 volume of chloroform/NaCl. After alkalization with NaOH, the alkaline aqueous phase was transferred into a siliconized glass tube, and the organic phase was re-extracted with methanol/NaCl/NaOH. The aqueous phases were combined, acidified, and extracted twice with chloroform. The organic phases were evaporated, and the dried lipids were dissolved in methanol/K2HPO4. The resolved lipids were derivatized with o-phthaldialdehyde. The derivatives were analyzed with a Merck-Hitachi LiChrom HPLC system (Merck-Hitachi, Darmstadt, Germany) using an RP 18 Kromasil column (Chromatographie Service GmbH, Langerwehe, Germany). Separation was done with a gradient of methanol/K2HPO4 (0.07 mol/liter). The recovery of S1P was calculated using dihydro-S1P as a standard (20Ruwisch L. Schafer-Korting M. Kleuser B. Naunyn. Schmiedeberg's Arch. Pharmacol. 2001; 363: 358-363Crossref PubMed Scopus (65) Google Scholar, 22Nofer J.R. van der Giet M. Tolle M. Wolinska I. von Wnuck Lipinski K. Baba H.A. Tietge U.J. Godecke A. Ishii I. Kleuser B. Schafers M. Fobker M. Zidek W. Assmann G. Chun J. Levkau B. J. Clin. Invest. 2004; 113: 569-581Crossref PubMed Scopus (587) Google Scholar). Angiogenesis Assay on Matrigel—Growth factor-reduced Matrigel matrix was purchased from BD Biosciences. Gels were allowed to polymerize in a 96-well plate for 30 min at 37 °C. HUVEC were seeded at 1 × 104/well and grown in endothelial cell medium supplemented with 1% fetal calf serum and without growth supplement for 18 h in a humidified 37 °C, 5% CO2 incubator. Tube formation was observed using a light microscope (Olympus BX51, Hamburg, Germany), and pictures were captured with a computer system and subjected to image processing using Image-Pro 4.5 image analysis software (CyberView Corp., Suwanee, GA). The extent of tube formation on matrix gels was expressed as total length of sprouts per captured area. Each control or test compound was assayed in duplicate, and the assays were performed three times. Statistical Analysis—Data are presented as mean ± S.D. unless indicated otherwise. Results were analyzed using Student’s t test, and statistical significance for all comparisons was assigned at p < 0.05. Mitogenic and Angiogenic Activities of Human Follicular Fluid—First, we examined the ability of native human follicular fluid to stimulate endothelial cell proliferation as determined by MTT assay. As shown in Fig. 1A, follicular fluid induced HUVEC proliferation at different concentrations tested (10–80 μl of follicular fluid/well, protein concentration, ∼47 g/liter). Basal endothelial proliferation was used as control and compared with proliferation after the addition of follicular fluid. Depending on the amount of follicular fluid added, the increases in proliferation varied from 25 to 60%. In order to characterize the temperature stability of mitogenic mediators, follicular fluid was subjected to incubation at different temperatures (from 25 to 100 °C) for 30 min. Denatured proteins were removed by centrifugation, and the supernatant was used for determination of growth factor concentration by immunoassay or mitogenic activity by proliferation assay. No detectable amounts of bFGF or VEGF were found in the follicular fluid after heat treatment (not shown). Interestingly, the addition of heat-treated follicular fluid to endothelial cells resulted in a marked increase in endothelial proliferation rate, and the heat stability could be demonstrated for up to 100 °C (Fig. 1B). The mitogenic effect of follicular fluid increased with rising temperature during pretreatment of follicular fluid and reached a maximum at 90 °C. This might reflect a temperature-dependent removal of antiangiogenic proteins by heat denaturation. To further characterize the contribution of bFGF and VEGF to mitogenic activity of follicular fluid, the growth rate of endothelial cells exposed to follicular fluid, in which bFGF and VEGF were neutralized with specific monoclonal antibodies, was determined. To achieve this, follicular fluid was incubated for 1 hour at room temperature with antibodies against bFGF and VEGF at an antibody/growth factor molar ratio of 1000:1. Concentrations of bFGF and VEGF in pooled native follicular fluid were 160 pg/ml and 3.6 ng/ml, respectively, and were reduced below the detection limit after pretreatment with antibodies. However, the mitogenic activity of follicular fluid under these experimental conditions was reduced only by 12 ± 5% (n = 3, not significant) and 29 ± 4% (n = 3, not significant), respectively, as compared with untreated follicular fluid (Fig. 1C), indicating that some unidentified residual angiogenic activity was present in follicular fluid after immunoneutralization. Although endothelial proliferation is critical for the formation of new vessels, it may be argued that proliferative effects alone are not sufficient for supporting effective angiogenesis. Therefore, we next sought to directly examine angiogenic effects of follicular fluid using an assay for new vessel formation. When placed on growth factor-reduced Matrigel in the absence of angiogenic factors or follicular fluid, HUVEC formed very few incomplete and narrow tube-like structures (Fig. 1D, Control). Untreated follicular fluid induced the development of expansive tubes organized by a high number of endothelial cells (Fig. 1D, FF). Similar although less pronounced effects were observed in the presence of heat-treated follicular fluid (ht-FF) and follicular fluid after immunoneutralization of bFGF or VEGF (Fig. 1D, ht-FF, FF + anti-bFGF, FF + anti-VEGF). Total sprouted area amounted to 458.3 ± 24 and 15,230 ± 238 pixels in the absence or presence of native follicular fluid (n = 3, p < 0.01), 7826 ± 84 pixels in the presence of heat-treated follicular fluid (n = 3; p < 0.01), and 13,773 ± 239 and 15,133 ± 832 pixels in the presence of follicular fluid pretreated with antibodies against VEGF and bFGF (n = 3; not significant), respectively. Collectively, these results point to the presence of a heat-stable mitogenic and angiogenic activity distinct from bFGF and VEGF in follicular fluid. Purification and Identification of the Mitogenic Activity of Human Follicular Fluid—Heat-stable mitogenic activity from human follicular fluid was partially purified in a two-step procedure. Heat treatment resulted in an approximately 2-fold increase of the relative mitogenic activity, whereas protein content decreased from 48.3 to 2.0 mg/ml. This indicates that the heat pretreatment is an effective initial step in the purification of mitogenic activity. Ion exchange chromatography on DEAE-Sepharose was applied as a second step. As indicated in Fig. 2A (arrows), three protein-rich fractions with retention times between 65 and 80 min could be eluted from the DEAE-Sepharose column using a linear NaCl gradient (0.1–0.4 mol/liter). All eluted fractions were subsequently pooled, desalted, and tested for their mitogenic activity in the MTT assay. As shown in Fig. 2B, the protein-rich DEAE fraction markedly increased endothelial cell proliferation. The subsequent SDS-PAGE of the mitogenically active fraction obtained by DEAE chromatography revealed five protein bands with molecular masses of approximately 14, 28, 43, 47, and 50 kDa (Fig. 2C). Dominant bands were excised from the gel, trypsinized, and subjected to the MALDI mass spectrometric analysis. The mass spectrograms of protein digests and the sequences of derived tryptic peptides are shown in Fig. 2D. Apolipoproteins A-I (SwissProt P02647) and A-IV (SwissProt P06727) were identified as major constituents of 28- and 43-kDa bands. Although a large part of the sequence was detected, signals for some terminal peptides were missing. Whereas the possibility cannot be entirely excluded that apolipoproteins present in follicular fluid are shorter, it is also possible that they were not detected with mass spectrometry due to suppression effects. In addition to bands corresponding to apoA-I and apoA-IV, several weak bands with lower molecular masses were observed that may represent the products of apolipoprotein cleavage occurring at aspartic acid residues at high temperature. Modifications of several amino acid residues were observed, such as the oxidation of methionine residues 164, 245, and 322 in apoA-IV and 118 and 154 in apoA-I. In addition, a large signal suggests ester formation at glutamic acid residue 96 of apoA-IV. Mitogenic Activity of HDL Isolated from Follicular Fluid—Because apoA-I and apoA-IV are major protein constituents of HDL, we assumed that the observed heat-stable mitogenic activity of human follicular fluid was induced by this lipoprotein fraction. As shown in Fig. 3A, FF-HDL induced proliferation of HUVEC in a dose-dependent manner with effective stimulation at a concentration of ∼0.4 mg/ml, which is close to the physiological level of HDL in follicular fluid (23Jaspard B. Fournier N. Vieitez G. Atger V. Barbaras R. Vieu C. Manent J. Chap H. Perret B. Collet X. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1605-1613Crossref PubMed Scopus (54) Google Scholar). Fig. 3B demonstrates that the heat-treated HDL displayed no reduction in the mitogenic activity as compared with the native FF-HDL. Furthermore, more than 90% of HDL estimated as apoA-I concentration could be recovered from heat-treated follicular fluid. We conclude that the mitogenic activity of FF-HDL is heat-stable. To further examine the contribution of HDL to the mitogenic activity exerted by follicular fluid, the effect of HDL depletion on follicular fluid-induced endothelial growth was investigated. The removal of an HDL fraction from follicular fluid by a discontinuous gradient centrifugation reduced its apoA-I content from 23.2 ± 0.05 to 4.8 ± 0.06 mg/dl (n = 4) and the HDL-cholesterol content from 12.2 ± 2.4 to 2.25 ± 1.5 mg/dl. Fig. 3C (inset) demonstrates a marked reduction of the electrophoretic HDL fraction after ultracentrifugation. Follicular fluid was previously demonstrated to contain two HDL subfractions, α and pre-β, characterized by distinct migration patterns in two-dimensional electrophoresis. As shown in Fig. 3D, the reduction of HDL content in the follicular fluid after ultracentrifugation was largely accounted by the depletion of phospholipid and cholesterol-rich α-HDL subfraction. The preferential removal of the α-HDL subfraction is also reflected by a disproportional reduction of HDL-cholesterol concentration in comparison with the apoA-I concentration in follicular fluid after ultracentrifugation. Fig. 3E demonstrates that HDL-depleted follicular fluid exhibited a markedly reduced ability to stimulate endothelial cell proliferation as compared with its native counterpart. HDL is known to be a complex lipoprotein fraction comprising several distinct protein and lipid entities. To discriminate which component of HDL accounts for its mitogenic activity, the endothelial proliferation-inducing effects of proteins or lipid fraction derived from HDL were investigated. As shown in Fig. 4A, purified apolipoproteins A-I and A-IV were not able to substitute the native FF-HDL in its ability to stimulate endothelial cell proliferation. Similarly, neither apoA-I complexed to phosphatidylcholine nor phosphatidylcholine-containing liposomes exerted any mitogenic activity. By contrast, the lipid fraction isolated from HDL exhibited a strong mitogenic effect toward endothelial cells. Both the mitogenic effects of FF-HDL and the lipid fraction isolated from FF-HDL were comparable with effects of HDL obtained from plasma. Recently, several lysophospholipids with potent mitogenic properties such as S1P were identified in the HDL fraction of human plasma. Therefore, we next examined whether follicular fluid and FF-HDL contain detectable amounts of S1P. As shown in Fig. 4B, S1P could be detected in both native follicular fluid and FF-HDL. The concentration of S1P in follicular fluid was estimated at 0.87 ± 0.11 ng/mg protein (n = 3; ∼170 nmol/liter). At this concentration, S1P was found to efficiently stimulate endothelial cell proliferation as determined by an MTT assay (Fig. 1C). Furthermore, the S1P content of the follicular fluid was considerably reduced after the removal of FF-HDL by performing discontinuous gradient centrifugation (Fig. 4D), indicating that this lysosphospholipid is associated with FF-HDL. To examine the contribution of S1P to mitogenic effects exerted by follicular fluid, we performed a digestion with alkaline phosphatase, which is known to degrade S1P (18Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7832) Google Scholar). As shown in Fig. 4E, alkaline phosphatase pretreatment of FF-HDL reduced the initially observed increase in endothelial cell proliferation as compared with the native FF-HDL. Contribution of Various Signal Transduction Pathways to Follicular Fluid-induced Proliferation—One consequence of the predicted role of S1P and FF-HDL in the follicular fluid-induced endothelial proliferation is that

Bovine anterior-pituitary microsomal fractions exhibit high-affinity, saturable and reversible binding of inositol 1,4,5-[32P]trisphosphate; 50% of the labelled ligand is displaced by 3.5 nM-inositol 1,4,5-trisphosphate. 0.5 microM-inositol 1,4-bisphosphate and 10 microM-ATP. Inositol 1,4,5-trisphosphate induces the release of Ca2+ from the microsomal vesicles (half-maximal effect at 290 nM), and its action is potentiated by inositol tetrakisphosphate (half-maximal effect at 4 microM).

The stimulation of luteinizing hormone (LH) release and cyclic GMP (cGMP) production in rat anterior pituitary cells by gonadotropin-releasing hormone (GnRH) are receptor mediated and calcium dependent, and have been shown to be accompanied by increased phospholipid turnover and arachidonic acid release. The incorporation of 32Pi into the total phospholipid fraction of pituitary gonadotrophs was significantly elevated by 10−8m GnRH, with specific increases in the labeling of phosphatidylinositol and phosphatidic acid (PA). Since PA acts as a calcium ionophore in several cell types, its effects upon calcium-mediated gonadotroph responses were compared with those elicited by GnRH. In rat pituitary gonadotrophs prepared by centrifugal elutriation, PA stimulated LH release and cGMP production by 9-fold and 5-fold, respectively. The stimulation of LH release by 30 μm PA was biphasic in its dependence on extracellular calcium concentration, rising from zero in the absence of calcium to a maximum of 10-fold at 0.5 mm Ca2+ and declining at higher calcium concentrations. In dose-response experiments, PA was 3-fold more potent at 0.5 mm Ca2+ than at 1.2 mm Ca2+. The cGMP response to PA in cultured gonadotrophs was also calcium dependent, and was progressively enhanced by increasing Ca2+ concentrations up to 1.5 mm. The ability of PA to stimulate both LH release and cGMP formation in a calcium-dependent manner suggests that endogenous PA formed in response to GnRH receptor activation could function as a Ca2+ ionophore in pituitary gonadotrophs, and may participate in the stimulation of gonadotroph responses by GnRH and its agonist analogs.

The effect of misoprostol, a PGE1 methyl analogue, on the pregnant human uterus was unknown at dosage levels normally used in the treatment of gastric and duodenal ulceration. Data from animal fertility and teratology studies suggested no activity at an anti-ulcer dosage level. In a double-blind placebo-controlled study, 300 patients (9.-12. week of gestation) were treated with two doses of misoprostol (study A: 2 X 400 micrograms; study B: 2 X 200 micrograms) or placebo during the evening before a legally permitted termination of first-trimester pregnancy. A partial or complete abortion occurred spontaneously in 11% of patients receiving misoprostol 2 X 400 micrograms, 9% of patients receiving misoprostol 2 X 200 micrograms and none of the patients receiving placebo. The incidence of vaginal bleedings (A: 45%, B: 34%), abdominal pain (A: 42%, B: 43%) and the softening of the cervix were all significantly increased by misoprostol treatment. These results show that the sensitivity of the human pregnant uterus to prostaglandin analogues cannot be reliably predicted from animal studies. Furthermore, misoprostol should not be used in human first-trimester pregnancy. The effect of misoprostol on second and third-trimester pregnancy (e.g. labour induction) is still unknown.

BackgroundExposure to bacterial antigens and other environmental factors in combination with a genetic susceptibility have been implicated in the etiology of inflammatory bowel disease (IBD). As certain perinatal circumstances, e.g., delivery by cesarean section, predispose to a different intestinal colonizations the aim of this analysis was to define a potential influence on the development of IBD in later life.

Abstract Objective To assess the accuracy of categorization of breast ultrasound findings based on scoring for malignancy using the sonographic breast imaging‐reporting and data system (BI‐RADS). Methods Breast ultrasound was performed in 2462 patients between 2001 and 2004 at our unit. Sonographic findings were scored using analog criteria as in BI‐RADS for breast ultrasound (mass shape, margin, orientation, posterior acoustic features, lesion boundary, echo pattern). Each lesion was described using these features and classified into categories 1 to 5 according to the BI‐RADS for breast ultrasound. Categorization and biopsy results were compared. Results In twenty‐two (0.9%) patients breast ultrasound could not be evaluated because of extreme density of tissue. Normal breast ultrasound belonging to Category 1 was found in 871 (35.4%) patients. Simple cysts classified as Category 2 were observed in 712 (28.9%) women. In 491 (19.9%) patients, apparently benign solid masses (Category 3) were found. Suspicious masses were observed in 225 (9.1%) women and masses highly suggestive of malignancy were found in 141 (5.7%) patients (Categories 4 and 5, respectively). Histological examinations were available from 84 (17.1%) masses that had been classified by BI‐RADS as Category 3, in 97 (43.1%) from Category 4 and 106 (75.2%) from Category 5. Accordingly, the rate of malignant findings was 1.2% ( n = 1) in Category 3, 17% ( n = 16) in Category 4 and 94% ( n = 100) in Category 5. Conclusion Scoring breast ultrasound findings for malignancy based on criteria used for BI‐RADS breast ultrasound has a high accuracy, comparable to that obtained by BI‐RADS for mammography. Copyright © 2008 ISUOG. Published by John Wiley &amp; Sons, Ltd.

The cell surface heparan sulfate proteoglycan syndecan-1 (CD138) modulates the activity of chemokines, cytokines, integrins, and other adhesion molecules which play important roles in the regulation of inflammation. We have previously shown that syndecan-1-deficient murine leukocytes display increased interactions with endothelial cells and increased diapedesis in vivo and in vitro. In this study, we demonstrate that syndecan-1 has an important function as a negative modulator in the murine contact allergy model of oxazolone-mediated delayed-type hypersensitivity (DTH). Following elicitation of the DTH response, syndecan-1-deficient mice showed an increase in leukocyte recruitment, resulting in an increased and prolonged edema formation. Expression of the cytokines TNF-alpha and IL-6 of the chemokines CCL5/RANTES and CCL-3/MIP-1alpha and of the adhesion molecule ICAM-1 were significantly increased in syndecan-1-deficient compared with wild-type mice. In wild-type mice, syndecan-1 mRNA and protein expression was reduced during the DTH response. The differentially increased adhesion of syndecan-1-deficient leukocytes to ICAM-1 was efficiently inhibited in vitro by CD18-blocking Abs, which emerges as one mechanistic explanation for the anti-inflammatory effects of syndecan-1. Collectively, our results show an important role of syndecan-1 in the contact DTH reaction, identifying syndecan-1 as a novel target in anti-inflammatory therapy.

Summary For most azoospermic men testicular sperm extraction ( TESE ) is the only treatment, however it presents challenges for the ART laboratory, as the retrieval of motile spermatozoa is difficult. In the absence of sperm movement no unequivocal distinction can be made between either dead or immotile, but vital spermatozoa. However, a single laser shot directed to the tip of the tail allows recognition of viability because the flagellum coils at the area of impact. To rank the quality and the maturity of oocytes, polarization microscopy can be used. The zona score and the visualization of the meiotic spindle correlate with implantation and pregnancy rates. We compared 65 TESE ‐ ICSI cycles of the years 2007 and 2008 (Group 1, G1) with 58 TESE ‐ ICSI cycles of the years 2009 and 2010 (Group 2, G2). Testicular spermatozoa were injected according to motility and morphology into selected oocytes. In G1 both, oocyte and spermatozoa were rated using light microscopy only, whereas in G2 the laser was used for sperm selection and the oocytes were rated by light and polarization microscopy. In G2 we enhanced our fertilization rate ( FR ) significantly in comparison to G1 (G1 42.1% vs. G2 52.7%, p &lt; 0.001). The fertilization rate with immotile, but vital spermatozoa improved significantly when applying laser‐based selection ( p = 0.006). The laser selection of immotile spermatozoa and the use of polarization microscopy can enhance the FR of TESE ‐ ICSI . No negative effect of the laser was seen on birth rates. The FR with immotile, but vital spermatozoa clearly benefits from laser selection and is a non‐hazardous and safe method for the selection of viable but immotile sperm. To our knowledge this is the first report using new technology creating novel endpoints for the analysis of spermatozoa and oocytes in TESE ‐ ICSI .

Polycystic ovary syndrome (PCOS) is a frequent heterogenic disorder with a familial background. Androgenic effects, determining the clinical features of the syndrome, are mediated by the androgen receptor (AR), whose activity is modulated by a genetic polymorphism. We investigated the role of the CAG repeat polymorphism of the androgen receptor in PCOS.In the infertility unit of a university clinic, 72 PCOS patients were compared with 179 ovulatory controls undergoing a standardized diagnostic work-up. The number of CAG repeats was determined by PCR, labelling with IR-800 and PAGE. X-chromosome inactivation was assessed by a methylation-sensitive assay.Compared to controls, PCOS patients displayed a shorter mean CAG repeat length, encoding for higher AR activity (P=0.001). CAG repeat length correlated inversely with oligomenorrhea, a central androgen dependent feature of the syndrome (P=0.005). In a binomial regression analysis including BMI, LH and free testosterone, CAG repeat length was identified as an independent risk factor for PCOS (P=0.002).The CAG repeat polymorphism could constitute one of the genetic factors modulating the syndrome's phenotype, contributing to its clinical heterogeneity and associated metabolic consequences.

Expression of the pluripotency factors SOX-2, OCT-4, KLF-4, and NANOG was analyzed by quantitative real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence microscopy in the endometrium, myometrium, and endometriotic tissue of 36 patients. Aberrant expression of SOX-2 may indicate a stem cell origin of endometriosis, whereas the presence of all progenitor markers in endometrial tissue marks the endometrium as a potential source for induced pluripotent stem cell generation. Expression of the pluripotency factors SOX-2, OCT-4, KLF-4, and NANOG was analyzed by quantitative real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence microscopy in the endometrium, myometrium, and endometriotic tissue of 36 patients. Aberrant expression of SOX-2 may indicate a stem cell origin of endometriosis, whereas the presence of all progenitor markers in endometrial tissue marks the endometrium as a potential source for induced pluripotent stem cell generation. Stem cells persist in a quiescent, slowly proliferating state in an environment called the stem cell niche (1Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. et al.Identification of the haematopoietic stem cell niche and control of the niche size.Nature. 2003; 425: 836-841Crossref PubMed Scopus (2365) Google Scholar). Transit amplifying cells derived from multipotent stem cells are characterized by their high proliferative potential, progressively acquiring differentiation markers and ultimately producing terminally differentiated cells (2Cervello I. Simon C. Somatic stem cells in the endometrium.Reprod Sci. 2009; 16: 200-205Crossref PubMed Scopus (27) Google Scholar). Aberrant stem cell function may contribute to the pathogenesis of endometriosis (3Gargett C.E. Uterine stem cells: what is the evidence?.Human Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (273) Google Scholar), the growth of endometrioid tissue outside the uterine cavity, causing dysmenorrhoea, dyspareunia, subfertility, or noncyclic pelvic pain (4Revised American Society for Reproductive Medicine classification of endometriosis: 1996.Fertil Steril. 1997; 67: 817-821Abstract Full Text PDF PubMed Scopus (2232) Google Scholar). Stem cells that are inappropriately shed during retrograde menstruation (5Sampson J.A. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity.Am J Obstet Gynecol. 1927; 14: 422-469Abstract Full Text PDF Google Scholar) or ectopically distributed through lymphovascular metastasis (6Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (257) Google Scholar) may contribute to the pathogenetic process, because their high proliferative potential promotes rapid clonal expansion (7Gargett C.E. Chan R.W. Endometrial stem/progenitor cells and proliferative disorders of the endometrium.Minerva Ginecol. 2006; 58: 511-526PubMed Google Scholar, 8Kim C.M. Oh Y.J. Cho S.H. Chung D.J. Hwang J.Y. Park K.H. et al.Increased telomerase activity and human telomerase reverse transcriptase mRNA expression in the endometrium of patients with endometriosis.Hum Reprod. 2007; 22: 843-849Crossref PubMed Scopus (33) Google Scholar). The monoclonal origin of some endometriotic lesions (3Gargett C.E. Uterine stem cells: what is the evidence?.Human Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (273) Google Scholar, 9Jimbo H. Hitomi Y. Yoshikawa H. Yano T. Momoeda M. Sakamoto A. et al.Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts. Am J Pathol. 1997; 150: 1173-1178Google Scholar), the long-term culturing properties of cell clones established from endometriotic lesions (10Tanaka M. Kyo S. Kanaya T. Yatabe N. Nakamura M. Maida Y. et al.Evidence of the monoclonal composition of human endometrial epithelial glands and mosaic pattern of clonal distribution in luminal epithelium.Am J Pathol. 2003; 163: 295-301Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and the isolation of progenitor cells with high differentiation potential from menstrual blood (11Meng X. Ichim T.E. Zhong J. Rogers A. Yin Z. Jackson J. et al.Endometrial regenerative cells: a novel stem cell population.J Transl Med. 2007; 5: 57Crossref PubMed Scopus (424) Google Scholar) support the stem cell hypothesis of endometriosis. Adenomyosis is caused by extensive endomyometrial invagination of the basal endometrium (12Ferenczy A. Pathophysiology of adenomyosis.Hum Reprod Update. 1998; 4: 312-322Crossref PubMed Scopus (376) Google Scholar). The stem cell niche may be altered in adenomyosis, because variations in the niche may promote smooth muscle differentiation, causing myometrial hyperplasia (13Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (374) Google Scholar).Numerous putative stem cell markers have been described (3Gargett C.E. Uterine stem cells: what is the evidence?.Human Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (273) Google Scholar, 8Kim C.M. Oh Y.J. Cho S.H. Chung D.J. Hwang J.Y. Park K.H. et al.Increased telomerase activity and human telomerase reverse transcriptase mRNA expression in the endometrium of patients with endometriosis.Hum Reprod. 2007; 22: 843-849Crossref PubMed Scopus (33) Google Scholar, 11Meng X. Ichim T.E. Zhong J. Rogers A. Yin Z. Jackson J. et al.Endometrial regenerative cells: a novel stem cell population.J Transl Med. 2007; 5: 57Crossref PubMed Scopus (424) Google Scholar, 13Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (374) Google Scholar, 14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar, 15Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14653) Google Scholar). Differentiated cells can be reprogrammed by the ectopic expression of specific transcription factors, including SOX-2, OCT-4, KLF-4, NANOG, and c-MYC, thus forming induced pluripotent stem (iPS) cells (15Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14653) Google Scholar, 16Kim J.B. Greber B. Arauzo-Bravo M.J. Meyer J. Park K.I. Zaehres H. et al.Direct reprogramming of human neural stem cells by OCT4.Nature. 2009; 461: 649-654Crossref PubMed Scopus (557) Google Scholar, 17Huangfu D. Osafune K. Maehr R. Guo W. Eijkelenboom A. Chen S. et al.Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and SOX2.Nat Biotechnol. 2008; 26: 1269-1275Crossref PubMed Scopus (1113) Google Scholar). SOX-2 regulates both embryonic and adult stem cell determination, differentiation, and proliferation (18Otsubo T. Akiyama Y. Yanagihara K. Yuasa Y. SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis.Br J Cancer. 2008; 98: 824-831Crossref PubMed Scopus (174) Google Scholar, 19Go M.J. Takenaka C. Ohgushia H. Forced expression of SOX2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities.Exp Cell Res. 2008; 314: 1147-1154Crossref PubMed Scopus (97) Google Scholar). In this study, we investigated whether SOX-2 and associated pluripotency markers are expressed in human endometrium, possibly marking early progenitor cells, and whether endometriosis and adenomyosis are associated with altered number and location of SOX-2–expressing stromal and epithelial progenitor cells.This study received institutional review board approval (Greb1IX), and written consent was provided by all patients. Tissue specimens (endometrial biopsies [n = 12] and adenomyotic lesions [n = 6] from hysterectomized uteri and endometriotic lesions [n = 18]) for immunohistochemical investigations were acquired between 2002 and 2006 at Münster University Hospital from 36 women aged 28–56 years (mean, 36 years) undergoing operation. Eighteen patients had endometriosis (9 ovarian, 6 rectovaginal, 3 peritoneal), 6 patients had hysterectomy because of adenomyosis, and our reference group comprised 12 patients (6 proliferative/6 secretory phase) undergoing hysterectomy because of myoma. After surgery, hemotoxylin-stained endometrial sections were assessed by experienced histopathologists referring to established histologic criteria (20Noyes R.W. Hertig A.T. Rock J. Dating the endometrial biopsy.Fertil Steril. 1950; 1: 3-25Crossref Google Scholar) and investigated by immunohistochemistry. Adenomyosis was diagnosed when endometrial glands and stroma were identified within the myometrium, with glands being present approximately 2.5 mm below the endometrium (21Zaloudek C. Hendrickson M.R. Ademyosis and adenomyoma.in: J Kurman Robert Blaustein’s pathology of the female genital tract. 5th ed. Springer, New York2002: 599-600Google Scholar). Specimens of eight additional patients with hysterectomies (aged 42–62 years; mean, 47 years) diagnosed with leiomyoma (n = 5), endometriosis (n = 1), or adenomyosis (n = 2) were analyzed by quantitative real-time PCR (qPCR). Three proliferative, three secretory, and two samples of unknown cycle phase were included. Fresh hysterectomy specimens were brought to the pathology laboratory on ice and opened by an experienced pathologist. One quarter of the endometrial surface was scraped with a scalpel to obtain a pure endometrial sample. The uterine wall was serially sectioned, and macroscopically normal myometrium from the outer half of the uterine wall was cut from the slices to serve as a pure myometrial sample. All samples were immediately snap-frozen, stored in liquid nitrogen, and studied by qPCR.Ribonucleic acid was isolated (RNeasy Mini kit; Qiagen, Hilden, Germany) including a DNAse digestion step, and reverse transcribed (First-Strand cDNA Synthesis System; Fermentas, St.Leon-Rot, Germany). Quantitative real-time PCR was performed on triplicates as previously described (14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar), utilizing exon-spanning TaqMan probes for human SOX-2 (Hs00415716_m1), NANOG (Hs02387400_g1), OCT-4 (Hs00742896_s1), KLF-4 (Hs00358836_m1), and 18S rRNA (Hs99999901_s1) (Applied Biosystems, Darmstadt, Germany). Relative quantification was performed using the 2-ΔΔCt method (22Livak K.J. Schmittgen T.D. Analyzing real-time PCR data by the comparative C(T) method.Nat Protoc. 2008; 3: 1101-1108Crossref PubMed Scopus (16736) Google Scholar), normalizing to 18S rRNA expression.Immunohistochemistry was performed as previously described (14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar): antigen retrieval was achieved by incubating slides in citrate buffer (pH 6.0; Dako, Carpenteria, CA) for 30 minutes in a steamer. Blocked paraffin sections were incubated with mouse anti-human SOX-2 monoclonal antibody (1:100; R&D Systems, Minneapolis, MN) for 16 hours at 4°C. In negative controls, primary antibodies were omitted. Primary antibodies were detected using anti-mouse EnVision (Dako) and 3-amino-9-ethylcarbazole (AEC)+ substrate, with hemalum counterstaining (Merck, Darmstadt, Germany). SOX-2–positive cells were quantified in a blinded manner on 168–1,033 visual fields (320 μm × 430 μm) per numerically coded slide at 320× magnification using a Zeiss Axiophot100 photomicroscope and Axiovision software (Zeiss, Göttingen, Germany) (14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar). Cells displaying either strong nuclear or cytoplasmic expression of SOX-2 were counted as positive. Individual sample data were expressed as numbers of SOX-2–positive cells per visual field. For immunofluorescence microscopy, paraffin-embedded tissue sections processed up to antigen retrieval (14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar) were blocked (10% Aurion BSAc; Dako) for 30 minutes and incubated with monoclonal mouse anti–SOX-2 (1:100; R&D Systems) and polyclonal rabbit anti-telomerase reverse transcriptase (Calbiochem, San Diego, CA) (1:250) antibodies overnight at 4°C. Omission of primary antibodies served as a negative control. Phosphate-buffered saline–washed samples were incubated with AlexaFluor 546–conjugated goat anti-mouse IgG and AlexaFluor 488–conjugated donkey anti-rabbit IgG (1:600; Molecular Probes, Eugene, OR) in the dark at room temperature for 1 hour, followed by 4’,6-diamidino-2-phenylindole staining (Sigma, St. Louis, MO). Glycerol-mounted slides were observed with a Leica DM LB fluorescence microscope equipped with a Leica DC300F camera (Leica Microsystems, Wetzlar, Germany). Images of different fluorescence channels were merged using Photoshop 8.0 software (Adobe, San Jose, CA).Statistical analysis was performed using SigmaStat 3.1 and SPSS 15 software (SPSS, Chicago, IL), using the Mann-Whitney rank sum test for immunohistochemical staining analysis and Student’s paired t-test for qPCR results. A P value of <.05 was considered statistically significant.Pluripotency marker expression was investigated by qPCR using complementary DNA prepared from matched endometrial and myometrial biopsies. SOX-2, NANOG, KLF-4, and OCT-4 messenger RNA (mRNA) were detectable in all patient tissues (Fig. 1A ). Comparison of the normalized cycle of threshold (Ct) values (22Livak K.J. Schmittgen T.D. Analyzing real-time PCR data by the comparative C(T) method.Nat Protoc. 2008; 3: 1101-1108Crossref PubMed Scopus (16736) Google Scholar) revealed that endometrial SOX-2 mRNA expression was 60 times lower than OCT-4 or NANOG expression and 27 times lower than KLF-4 expression (not shown). Endometrial OCT-4 expression was significantly increased, 9.8-fold over myometrial expression, whereas NANOG expression was 2.7-fold increased (nonsignificant). In contrast, endometrial SOX-2 expression was significantly decreased, 0.51-fold compared with myometrial expression, whereas KLF-4 expression was decreased 0.62-fold (nonsignificant) (Fig. 1A).Immunofluorescence microscopy revealed perinuclear, punctate staining, or cytoplasmic staining patterns of stromal SOX-2, which colocalized with telomerase (Fig. 1B). Investigating 36 patient tissues by immunohistochemistry, nuclear and/or cytoplasmic staining for SOX-2 was detected in single stromal cells of proliferative-phase endometrium (Fig. 1C), cell groups in endometriotic glands (Fig. 1D), and endometriotic stroma (Fig. 1E) and as a diffuse cytoplasmic staining of endometrial glands in adenomyosis tissue (Fig. 1F). Frequently, SOX-2–positive cells showed a perivascular localization (Fig. 1G). SOX-2–expressing stromal cell numbers were significantly increased by >100% during the proliferative compared with the secretory phase (P<.05) (Fig. 1H). Glandular SOX-2 expression did not significantly differ between cycle phases (Fig. 1H), and no differences were observed in adenomyosis tissue compared with healthy endometrium (Fig. 1H and I). However, in endometriotic tissue, SOX-2–positive stromal cell numbers were significantly increased by 68% compared with secretory- but not proliferative-phase endometrium of healthy donors (Fig. 1H). Glandular SOX-2 expression was heterogeneous and did not significantly differ between all investigated entities (Fig. 1I).Our study demonstrates expression of the pluripotency factors SOX-2, OCT-4, KLF-4, and NANOG in human endometrium, myometrium, and endometriotic tissues. SOX-2 colocalized with telomerase, whose activity is associated with the immortality of embryonic stem cells and (endometrial) carcinoma cells (8Kim C.M. Oh Y.J. Cho S.H. Chung D.J. Hwang J.Y. Park K.H. et al.Increased telomerase activity and human telomerase reverse transcriptase mRNA expression in the endometrium of patients with endometriosis.Hum Reprod. 2007; 22: 843-849Crossref PubMed Scopus (33) Google Scholar, 14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar, 23Kyo S. Masutomi K. Maida Y. Kanaya T. Yatabe N. Nakamura M. et al.Significance of immunological detection of human telomerase reverse transcriptase: re-evaluation of expression and localization of human telomerase reverse transcriptase.Am J Pathol. 2003; 163: 859-867Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), emphasizing the stem cell character of SOX-2–positive cells. A potential caveat is associated with the lack of nuclear immuno-fluorescence staining of SOX-2 and telomerase reverse transcriptase, which may indicate absence of regulatory activity in the nucleus or may represent nucleocytoplasmic shuttling of the telomerase holoenzyme during the assembly process (23Kyo S. Masutomi K. Maida Y. Kanaya T. Yatabe N. Nakamura M. et al.Significance of immunological detection of human telomerase reverse transcriptase: re-evaluation of expression and localization of human telomerase reverse transcriptase.Am J Pathol. 2003; 163: 859-867Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).SOX-2–positive cells were frequently found in a perivascular location, albeit in lower quantities compared with the adult stem cell markers CD146, STRO-1, and CD90, which show a more widespread perivascular staining pattern (24Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (415) Google Scholar, 25Schwab K.E. Hutchinson P. Gargett C.E. Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells.Hum Reprod. 2008; 23: 934-943Crossref PubMed Scopus (165) Google Scholar). These findings are in agreement with the identification of bone marrow–derived mesenchymal stem cells populating the endometrium, and with the concept of lymphovascular metastasis as a pathogenetic route for endometriosis (6Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (257) Google Scholar, 24Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (415) Google Scholar).Reprogramming of differentiated cells to an embryonic stem cell–like state by forced expression of OCT-4, SOX-2, KLF-4, and c-MYC (15Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14653) Google Scholar) can be more easily induced in cells already expressing some of these factors (16Kim J.B. Greber B. Arauzo-Bravo M.J. Meyer J. Park K.I. Zaehres H. et al.Direct reprogramming of human neural stem cells by OCT4.Nature. 2009; 461: 649-654Crossref PubMed Scopus (557) Google Scholar, 17Huangfu D. Osafune K. Maehr R. Guo W. Eijkelenboom A. Chen S. et al.Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and SOX2.Nat Biotechnol. 2008; 26: 1269-1275Crossref PubMed Scopus (1113) Google Scholar). The novel finding of an expression of SOX-2, OCT-4, KLF-4, and NANOG marks the endometrium as an attractive potential source for the generation of iPS cells. Further research needs to establish whether all four factors are expressed by the same putative endometrial stem cell as a prerequisite for applying this technology. Because human endometrium can be obtained by low-invasive techniques, the generation of autologous iPS cells from endometrium may represent a promising future perspective.SOX-2–positive stroma cell numbers were significantly increased in the proliferative phase. Reflecting SOX-2 functions in cell cycle progression and mitogenic signaling (19Go M.J. Takenaka C. Ohgushia H. Forced expression of SOX2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities.Exp Cell Res. 2008; 314: 1147-1154Crossref PubMed Scopus (97) Google Scholar), this supports the stem cell concept of cyclic endometrial regeneration (2Cervello I. Simon C. Somatic stem cells in the endometrium.Reprod Sci. 2009; 16: 200-205Crossref PubMed Scopus (27) Google Scholar, 3Gargett C.E. Uterine stem cells: what is the evidence?.Human Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (273) Google Scholar, 6Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (257) Google Scholar, 7Gargett C.E. Chan R.W. Endometrial stem/progenitor cells and proliferative disorders of the endometrium.Minerva Ginecol. 2006; 58: 511-526PubMed Google Scholar). Future investigations of endometrial samples according to menstrual cycle phase may be worthwhile. Significantly increased numbers of SOX-2–positive stroma cells in endometriotic lesions compared with secretory endometrium support the stem cell concept of endometriosis. Future studies need to address the full dif-ferentiation potential of endometrial SOX-2–positive cells, to provide formal proof of their stem cell properties. Furthermore, histopathologic investigations of the predictive value of stromal SOX-2 expression in expanded patient collectives, which need to be complemented by functional studies, are warranted to firmly establish its clinical relevance for the pathogenesis of endometriosis. Stem cells persist in a quiescent, slowly proliferating state in an environment called the stem cell niche (1Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. et al.Identification of the haematopoietic stem cell niche and control of the niche size.Nature. 2003; 425: 836-841Crossref PubMed Scopus (2365) Google Scholar). Transit amplifying cells derived from multipotent stem cells are characterized by their high proliferative potential, progressively acquiring differentiation markers and ultimately producing terminally differentiated cells (2Cervello I. Simon C. Somatic stem cells in the endometrium.Reprod Sci. 2009; 16: 200-205Crossref PubMed Scopus (27) Google Scholar). Aberrant stem cell function may contribute to the pathogenesis of endometriosis (3Gargett C.E. Uterine stem cells: what is the evidence?.Human Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (273) Google Scholar), the growth of endometrioid tissue outside the uterine cavity, causing dysmenorrhoea, dyspareunia, subfertility, or noncyclic pelvic pain (4Revised American Society for Reproductive Medicine classification of endometriosis: 1996.Fertil Steril. 1997; 67: 817-821Abstract Full Text PDF PubMed Scopus (2232) Google Scholar). Stem cells that are inappropriately shed during retrograde menstruation (5Sampson J.A. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity.Am J Obstet Gynecol. 1927; 14: 422-469Abstract Full Text PDF Google Scholar) or ectopically distributed through lymphovascular metastasis (6Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (257) Google Scholar) may contribute to the pathogenetic process, because their high proliferative potential promotes rapid clonal expansion (7Gargett C.E. Chan R.W. Endometrial stem/progenitor cells and proliferative disorders of the endometrium.Minerva Ginecol. 2006; 58: 511-526PubMed Google Scholar, 8Kim C.M. Oh Y.J. Cho S.H. Chung D.J. Hwang J.Y. Park K.H. et al.Increased telomerase activity and human telomerase reverse transcriptase mRNA expression in the endometrium of patients with endometriosis.Hum Reprod. 2007; 22: 843-849Crossref PubMed Scopus (33) Google Scholar). The monoclonal origin of some endometriotic lesions (3Gargett C.E. Uterine stem cells: what is the evidence?.Human Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (273) Google Scholar, 9Jimbo H. Hitomi Y. Yoshikawa H. Yano T. Momoeda M. Sakamoto A. et al.Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts. Am J Pathol. 1997; 150: 1173-1178Google Scholar), the long-term culturing properties of cell clones established from endometriotic lesions (10Tanaka M. Kyo S. Kanaya T. Yatabe N. Nakamura M. Maida Y. et al.Evidence of the monoclonal composition of human endometrial epithelial glands and mosaic pattern of clonal distribution in luminal epithelium.Am J Pathol. 2003; 163: 295-301Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and the isolation of progenitor cells with high differentiation potential from menstrual blood (11Meng X. Ichim T.E. Zhong J. Rogers A. Yin Z. Jackson J. et al.Endometrial regenerative cells: a novel stem cell population.J Transl Med. 2007; 5: 57Crossref PubMed Scopus (424) Google Scholar) support the stem cell hypothesis of endometriosis. Adenomyosis is caused by extensive endomyometrial invagination of the basal endometrium (12Ferenczy A. Pathophysiology of adenomyosis.Hum Reprod Update. 1998; 4: 312-322Crossref PubMed Scopus (376) Google Scholar). The stem cell niche may be altered in adenomyosis, because variations in the niche may promote smooth muscle differentiation, causing myometrial hyperplasia (13Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (374) Google Scholar). Numerous putative stem cell markers have been described (3Gargett C.E. Uterine stem cells: what is the evidence?.Human Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (273) Google Scholar, 8Kim C.M. Oh Y.J. Cho S.H. Chung D.J. Hwang J.Y. Park K.H. et al.Increased telomerase activity and human telomerase reverse transcriptase mRNA expression in the endometrium of patients with endometriosis.Hum Reprod. 2007; 22: 843-849Crossref PubMed Scopus (33) Google Scholar, 11Meng X. Ichim T.E. Zhong J. Rogers A. Yin Z. Jackson J. et al.Endometrial regenerative cells: a novel stem cell population.J Transl Med. 2007; 5: 57Crossref PubMed Scopus (424) Google Scholar, 13Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (374) Google Scholar, 14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar, 15Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14653) Google Scholar). Differentiated cells can be reprogrammed by the ectopic expression of specific transcription factors, including SOX-2, OCT-4, KLF-4, NANOG, and c-MYC, thus forming induced pluripotent stem (iPS) cells (15Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14653) Google Scholar, 16Kim J.B. Greber B. Arauzo-Bravo M.J. Meyer J. Park K.I. Zaehres H. et al.Direct reprogramming of human neural stem cells by OCT4.Nature. 2009; 461: 649-654Crossref PubMed Scopus (557) Google Scholar, 17Huangfu D. Osafune K. Maehr R. Guo W. Eijkelenboom A. Chen S. et al.Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and SOX2.Nat Biotechnol. 2008; 26: 1269-1275Crossref PubMed Scopus (1113) Google Scholar). SOX-2 regulates both embryonic and adult stem cell determination, differentiation, and proliferation (18Otsubo T. Akiyama Y. Yanagihara K. Yuasa Y. SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis.Br J Cancer. 2008; 98: 824-831Crossref PubMed Scopus (174) Google Scholar, 19Go M.J. Takenaka C. Ohgushia H. Forced expression of SOX2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities.Exp Cell Res. 2008; 314: 1147-1154Crossref PubMed Scopus (97) Google Scholar). In this study, we investigated whether SOX-2 and associated pluripotency markers are expressed in human endometrium, possibly marking early progenitor cells, and whether endometriosis and adenomyosis are associated with altered number and location of SOX-2–expressing stromal and epithelial progenitor cells. This study received institutional review board approval (Greb1IX), and written consent was provided by all patients. Tissue specimens (endometrial biopsies [n = 12] and adenomyotic lesions [n = 6] from hysterectomized uteri and endometriotic lesions [n = 18]) for immunohistochemical investigations were acquired between 2002 and 2006 at Münster University Hospital from 36 women aged 28–56 years (mean, 36 years) undergoing operation. Eighteen patients had endometriosis (9 ovarian, 6 rectovaginal, 3 peritoneal), 6 patients had hysterectomy because of adenomyosis, and our reference group comprised 12 patients (6 proliferative/6 secretory phase) undergoing hysterectomy because of myoma. After surgery, hemotoxylin-stained endometrial sections were assessed by experienced histopathologists referring to established histologic criteria (20Noyes R.W. Hertig A.T. Rock J. Dating the endometrial biopsy.Fertil Steril. 1950; 1: 3-25Crossref Google Scholar) and investigated by immunohistochemistry. Adenomyosis was diagnosed when endometrial glands and stroma were identified within the myometrium, with glands being present approximately 2.5 mm below the endometrium (21Zaloudek C. Hendrickson M.R. Ademyosis and adenomyoma.in: J Kurman Robert Blaustein’s pathology of the female genital tract. 5th ed. Springer, New York2002: 599-600Google Scholar). Specimens of eight additional patients with hysterectomies (aged 42–62 years; mean, 47 years) diagnosed with leiomyoma (n = 5), endometriosis (n = 1), or adenomyosis (n = 2) were analyzed by quantitative real-time PCR (qPCR). Three proliferative, three secretory, and two samples of unknown cycle phase were included. Fresh hysterectomy specimens were brought to the pathology laboratory on ice and opened by an experienced pathologist. One quarter of the endometrial surface was scraped with a scalpel to obtain a pure endometrial sample. The uterine wall was serially sectioned, and macroscopically normal myometrium from the outer half of the uterine wall was cut from the slices to serve as a pure myometrial sample. All samples were immediately snap-frozen, stored in liquid nitrogen, and studied by qPCR. Ribonucleic acid was isolated (RNeasy Mini kit; Qiagen, Hilden, Germany) including a DNAse digestion step, and reverse transcribed (First-Strand cDNA Synthesis System; Fermentas, St.Leon-Rot, Germany). Quantitative real-time PCR was performed on triplicates as previously described (14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar), utilizing exon-spanning TaqMan probes for human SOX-2 (Hs00415716_m1), NANOG (Hs02387400_g1), OCT-4 (Hs00742896_s1), KLF-4 (Hs00358836_m1), and 18S rRNA (Hs99999901_s1) (Applied Biosystems, Darmstadt, Germany). Relative quantification was performed using the 2-ΔΔCt method (22Livak K.J. Schmittgen T.D. Analyzing real-time PCR data by the comparative C(T) method.Nat Protoc. 2008; 3: 1101-1108Crossref PubMed Scopus (16736) Google Scholar), normalizing to 18S rRNA expression. Immunohistochemistry was performed as previously described (14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar): antigen retrieval was achieved by incubating slides in citrate buffer (pH 6.0; Dako, Carpenteria, CA) for 30 minutes in a steamer. Blocked paraffin sections were incubated with mouse anti-human SOX-2 monoclonal antibody (1:100; R&D Systems, Minneapolis, MN) for 16 hours at 4°C. In negative controls, primary antibodies were omitted. Primary antibodies were detected using anti-mouse EnVision (Dako) and 3-amino-9-ethylcarbazole (AEC)+ substrate, with hemalum counterstaining (Merck, Darmstadt, Germany). SOX-2–positive cells were quantified in a blinded manner on 168–1,033 visual fields (320 μm × 430 μm) per numerically coded slide at 320× magnification using a Zeiss Axiophot100 photomicroscope and Axiovision software (Zeiss, Göttingen, Germany) (14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar). Cells displaying either strong nuclear or cytoplasmic expression of SOX-2 were counted as positive. Individual sample data were expressed as numbers of SOX-2–positive cells per visual field. For immunofluorescence microscopy, paraffin-embedded tissue sections processed up to antigen retrieval (14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar) were blocked (10% Aurion BSAc; Dako) for 30 minutes and incubated with monoclonal mouse anti–SOX-2 (1:100; R&D Systems) and polyclonal rabbit anti-telomerase reverse transcriptase (Calbiochem, San Diego, CA) (1:250) antibodies overnight at 4°C. Omission of primary antibodies served as a negative control. Phosphate-buffered saline–washed samples were incubated with AlexaFluor 546–conjugated goat anti-mouse IgG and AlexaFluor 488–conjugated donkey anti-rabbit IgG (1:600; Molecular Probes, Eugene, OR) in the dark at room temperature for 1 hour, followed by 4’,6-diamidino-2-phenylindole staining (Sigma, St. Louis, MO). Glycerol-mounted slides were observed with a Leica DM LB fluorescence microscope equipped with a Leica DC300F camera (Leica Microsystems, Wetzlar, Germany). Images of different fluorescence channels were merged using Photoshop 8.0 software (Adobe, San Jose, CA). Statistical analysis was performed using SigmaStat 3.1 and SPSS 15 software (SPSS, Chicago, IL), using the Mann-Whitney rank sum test for immunohistochemical staining analysis and Student’s paired t-test for qPCR results. A P value of <.05 was considered statistically significant. Pluripotency marker expression was investigated by qPCR using complementary DNA prepared from matched endometrial and myometrial biopsies. SOX-2, NANOG, KLF-4, and OCT-4 messenger RNA (mRNA) were detectable in all patient tissues (Fig. 1A ). Comparison of the normalized cycle of threshold (Ct) values (22Livak K.J. Schmittgen T.D. Analyzing real-time PCR data by the comparative C(T) method.Nat Protoc. 2008; 3: 1101-1108Crossref PubMed Scopus (16736) Google Scholar) revealed that endometrial SOX-2 mRNA expression was 60 times lower than OCT-4 or NANOG expression and 27 times lower than KLF-4 expression (not shown). Endometrial OCT-4 expression was significantly increased, 9.8-fold over myometrial expression, whereas NANOG expression was 2.7-fold increased (nonsignificant). In contrast, endometrial SOX-2 expression was significantly decreased, 0.51-fold compared with myometrial expression, whereas KLF-4 expression was decreased 0.62-fold (nonsignificant) (Fig. 1A). Immunofluorescence microscopy revealed perinuclear, punctate staining, or cytoplasmic staining patterns of stromal SOX-2, which colocalized with telomerase (Fig. 1B). Investigating 36 patient tissues by immunohistochemistry, nuclear and/or cytoplasmic staining for SOX-2 was detected in single stromal cells of proliferative-phase endometrium (Fig. 1C), cell groups in endometriotic glands (Fig. 1D), and endometriotic stroma (Fig. 1E) and as a diffuse cytoplasmic staining of endometrial glands in adenomyosis tissue (Fig. 1F). Frequently, SOX-2–positive cells showed a perivascular localization (Fig. 1G). SOX-2–expressing stromal cell numbers were significantly increased by >100% during the proliferative compared with the secretory phase (P<.05) (Fig. 1H). Glandular SOX-2 expression did not significantly differ between cycle phases (Fig. 1H), and no differences were observed in adenomyosis tissue compared with healthy endometrium (Fig. 1H and I). However, in endometriotic tissue, SOX-2–positive stromal cell numbers were significantly increased by 68% compared with secretory- but not proliferative-phase endometrium of healthy donors (Fig. 1H). Glandular SOX-2 expression was heterogeneous and did not significantly differ between all investigated entities (Fig. 1I). Our study demonstrates expression of the pluripotency factors SOX-2, OCT-4, KLF-4, and NANOG in human endometrium, myometrium, and endometriotic tissues. SOX-2 colocalized with telomerase, whose activity is associated with the immortality of embryonic stem cells and (endometrial) carcinoma cells (8Kim C.M. Oh Y.J. Cho S.H. Chung D.J. Hwang J.Y. Park K.H. et al.Increased telomerase activity and human telomerase reverse transcriptase mRNA expression in the endometrium of patients with endometriosis.Hum Reprod. 2007; 22: 843-849Crossref PubMed Scopus (33) Google Scholar, 14Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (162) Google Scholar, 23Kyo S. Masutomi K. Maida Y. Kanaya T. Yatabe N. Nakamura M. et al.Significance of immunological detection of human telomerase reverse transcriptase: re-evaluation of expression and localization of human telomerase reverse transcriptase.Am J Pathol. 2003; 163: 859-867Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), emphasizing the stem cell character of SOX-2–positive cells. A potential caveat is associated with the lack of nuclear immuno-fluorescence staining of SOX-2 and telomerase reverse transcriptase, which may indicate absence of regulatory activity in the nucleus or may represent nucleocytoplasmic shuttling of the telomerase holoenzyme during the assembly process (23Kyo S. Masutomi K. Maida Y. Kanaya T. Yatabe N. Nakamura M. et al.Significance of immunological detection of human telomerase reverse transcriptase: re-evaluation of expression and localization of human telomerase reverse transcriptase.Am J Pathol. 2003; 163: 859-867Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). SOX-2–positive cells were frequently found in a perivascular location, albeit in lower quantities compared with the adult stem cell markers CD146, STRO-1, and CD90, which show a more widespread perivascular staining pattern (24Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (415) Google Scholar, 25Schwab K.E. Hutchinson P. Gargett C.E. Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells.Hum Reprod. 2008; 23: 934-943Crossref PubMed Scopus (165) Google Scholar). These findings are in agreement with the identification of bone marrow–derived mesenchymal stem cells populating the endometrium, and with the concept of lymphovascular metastasis as a pathogenetic route for endometriosis (6Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (257) Google Scholar, 24Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (415) Google Scholar). Reprogramming of differentiated cells to an embryonic stem cell–like state by forced expression of OCT-4, SOX-2, KLF-4, and c-MYC (15Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14653) Google Scholar) can be more easily induced in cells already expressing some of these factors (16Kim J.B. Greber B. Arauzo-Bravo M.J. Meyer J. Park K.I. Zaehres H. et al.Direct reprogramming of human neural stem cells by OCT4.Nature. 2009; 461: 649-654Crossref PubMed Scopus (557) Google Scholar, 17Huangfu D. Osafune K. Maehr R. Guo W. Eijkelenboom A. Chen S. et al.Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and SOX2.Nat Biotechnol. 2008; 26: 1269-1275Crossref PubMed Scopus (1113) Google Scholar). The novel finding of an expression of SOX-2, OCT-4, KLF-4, and NANOG marks the endometrium as an attractive potential source for the generation of iPS cells. Further research needs to establish whether all four factors are expressed by the same putative endometrial stem cell as a prerequisite for applying this technology. Because human endometrium can be obtained by low-invasive techniques, the generation of autologous iPS cells from endometrium may represent a promising future perspective. SOX-2–positive stroma cell numbers were significantly increased in the proliferative phase. Reflecting SOX-2 functions in cell cycle progression and mitogenic signaling (19Go M.J. Takenaka C. Ohgushia H. Forced expression of SOX2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities.Exp Cell Res. 2008; 314: 1147-1154Crossref PubMed Scopus (97) Google Scholar), this supports the stem cell concept of cyclic endometrial regeneration (2Cervello I. Simon C. Somatic stem cells in the endometrium.Reprod Sci. 2009; 16: 200-205Crossref PubMed Scopus (27) Google Scholar, 3Gargett C.E. Uterine stem cells: what is the evidence?.Human Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (273) Google Scholar, 6Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (257) Google Scholar, 7Gargett C.E. Chan R.W. Endometrial stem/progenitor cells and proliferative disorders of the endometrium.Minerva Ginecol. 2006; 58: 511-526PubMed Google Scholar). Future investigations of endometrial samples according to menstrual cycle phase may be worthwhile. Significantly increased numbers of SOX-2–positive stroma cells in endometriotic lesions compared with secretory endometrium support the stem cell concept of endometriosis. Future studies need to address the full dif-ferentiation potential of endometrial SOX-2–positive cells, to provide formal proof of their stem cell properties. Furthermore, histopathologic investigations of the predictive value of stromal SOX-2 expression in expanded patient collectives, which need to be complemented by functional studies, are warranted to firmly establish its clinical relevance for the pathogenesis of endometriosis. The authors thank Barbara Kloke, Ruth Goez, and Birgit Pers for expert technical assistance; and Tobias Cantz, M.D., and participants of the First North Rhine Westphalian Stem Cell School for stimulating discussions.

Triple negative breast cancer (TNBC) is characterized by lack of expression of both estrogen and progesterone receptor as well as lack of overexpression or amplification of HER2. Despite an increased probability of response to chemotherapy, many patients resistant to current chemotherapy regimens suffer from a worse prognosis compared to other breast cancer subtypes. However, molecular determinants of response to chemotherapy specific to TNBC remain largely unknown. Thus, there is a high demand for biomarkers potentially stratifying triple negative breast cancer patients for neoadjuvant chemotherapies or alternative therapies. In order to identify genes correlating with both the triple negative breast cancer subtype as well as response to neoadjuvant chemotherapy we employed publicly available gene expression profiles of patients, which had received neoadjuvant chemotherapy. Analysis of tissue microarrays as well as breast cancer cell lines revealed correlation to the triple negative breast cancer subtype. Subsequently, effects of siRNA-mediated knockdown on response to standard chemotherapeutic agents as well as radiation therapy were analyzed. Additionally, we evaluated the molecular mechanisms by which SFRP1 alters the carcinogenic properties of breast cancer cells. SFRP1 was identified as being significantly overexpressed in TNBC compared to other breast cancer subtypes. Additionally, SFRP1 expression is significantly correlated with an increased probability of positive response to neoadjuvant chemotherapy. Knockdown of SFRP1 in triple negative breast cancer cells renders the cells more resistant to standard chemotherapy. Moreover, tumorigenic properties of the cells are modified by knockdown, as shown by both migration or invasion capacity as well reduced apoptotic events. Surprisingly, we found that these effects do not rely on Wnt signaling. Furthermore, we show that pro-apoptotic as well as migratory pathways are differentially regulated after SFRP1 knockdown. We could firstly show that SFRP1 strongly correlates with the triple negative breast cancer subtype and secondly, that SFRP1 might be used as a marker stratifying patients to positively respond to neoadjuvant chemotherapy. The mechanisms by which tumor suppressor SFRP1 influences carcinogenic properties of cancer cells do not rely on Wnt signaling, thereby demonstrating the complexity of tumor associated signaling pathways.

Abstract Summary The first German interdisciplinary S3-guideline on the diagnosis, therapy and follow-up of patients with endometrial cancer was published in April 2018. Funded by German Cancer Aid as part of an Oncology Guidelines Program, the lead coordinators of the guideline were the German Society of Gynecology and Obstetrics (DGGG) and the Gynecological Oncology Working Group (AGO) of the German Cancer Society (DKG). Purpose Using evidence-based, risk-adapted therapy to treat low-risk women with endometrial cancer avoids unnecessarily radical surgery and non-useful adjuvant radiotherapy and/or chemotherapy. This can significantly reduce therapy-induced morbidity and improve the patientʼs quality of life as well as avoiding unnecessary costs. For women with endometrial cancer and a high risk of recurrence, the guideline defines the optimal extent of surgical radicality together with the appropriate chemotherapy and/or adjuvant radiotherapy if required. An evidence-based optimal use of different therapeutic modalities should improve the survival rates and quality of life of these patients. This S3-guideline on endometrial cancer is intended as a basis for certified gynecological cancer centers. The aim is that the quality indicators established in this guideline will be incorporated in the certification processes of these centers. Methods The guideline was compiled in accordance with the requirements for S3-level guidelines. This includes, in the first instance, the adaptation of source guidelines selected using the DELBI instrument for appraising guidelines. Other consulted sources included reviews of evidence, which were compiled from literature selected during systematic searches of literature databases using the PICO scheme. In addition, an external biostatistics institute was commissioned to carry out a systematic search and assessment of the literature for one part of the guideline. Identified materials were used by the interdisciplinary working groups to develop suggestions for Recommendations and Statements, which were then subsequently modified during structured consensus conferences and/or additionally amended online using the DELPHI method, with consent between members achieved online. The guideline report is freely available online. Recommendations Part 2 of this short version of the guideline presents recommendations for the therapy of endometrial cancer including precancers and early endometrial cancer as well as recommendations on palliative medicine, psycho-oncology, rehabilitation, patient information and healthcare facilities to treat endometrial cancer. The management of precancers of early endometrial precancerous conditions including fertility-preserving strategies is presented. The concept used for surgical primary therapy of endometrial cancer is described. Radiotherapy and adjuvant medical therapy to treat endometrial cancer and uterine carcinosarcomas are described. Recommendations are given for the follow-up care of endometrial cancer, recurrence and metastasis. Palliative medicine, psycho-oncology including psychosocial care, and patient information and rehabilitation are presented. Finally, the care algorithm and quality assurance steps for the diagnosis, therapy and follow-up of patients with endometrial cancer are outlined.

The therapeutic potential of Musashi (MSI) RNA-binding proteins, important stemness-associated gene expression regulators, remains insufficiently understood in breast cancer. This study identifies the interplay between MSI protein expression, stem cell characteristics, radioresistance, cell invasiveness and migration. MSI-1, MSI-2 and Notch pathway elements were investigated via quantitative polymerase chain reaction (qPCR) in 19 triple-negative breast cancer samples. Measurements were repeated in MDA-MB-231 cells after MSI-1 and -2 siRNA-mediated double knockdown, with further experiments performed after MSI silencing. Flow cytometry helped quantify expression of CD44 and leukemia inhibitory factor receptor (LIFR), changes in apoptosis and cell cycle progression. Proliferation and irradiation-induced effects were assessed using colony formation assays. Radiation-related proteins were investigated via Western blots. Finally, cell invasion assays and digital holographic microscopy for cell migration were performed. MSI proteins showed strong correlations with Notch pathway elements. MSI knockdown resulted in reduction of stem cell marker expression, cell cycle progression and proliferation, while increasing apoptosis. Cells were radiosensitized as radioresistance-conferring proteins were downregulated. However, MSI-silencing-mediated LIFR downregulation resulted in enhanced cell invasion and migration. We conclude that, while MSI knockdown results in several therapeutically desirable consequences, enhanced invasion and migration need to be counteracted before knockdown advantages can be fully exploited.

Genitourinary symptoms, such as vaginal dryness and pain with sex, are commonly experienced by postmenopausal women. Comparing treatments for these genitourinary symptoms are restricted by the use of different outcome measures in clinical trials and the omission of outcomes, which may be relevant to women. The aim of this project was to develop a Core Outcome Set (COS) to be reported in clinical trials of treatments for genitourinary symptoms associated with menopause.We performed a systematic review of randomized controlled trials of treatments for genitourinary symptoms associated with menopause and extracted their outcomes. This list was refined and entered into a two-round modified Delphi survey, which was open to clinicians, researchers, and postmenopausal women from November 2019 to March 2020. Outcomes were scored on a nine-point scale from "not important" to "critically important." The final COS was determined following two international consensus meetings.A total of 26 unique outcomes were included in the Delphi process, which was completed by 227 participants of whom 58% were postmenopausal women, 34% clinicians, and 8% researchers. Predefined thresholds were applied to the Delphi scores to categorize outcomes by importance, which informed the e consensus meetings, attended by 43 participants from 21 countries. The final COS includes eight outcomes: (1) pain with sex, (2) vulvovaginal dryness, (3) vulvovaginal discomfort or irritation, (4) discomfort or pain when urinating, (5) change in most bothersome symptom, (6) distress, bother or interference of genitourinary symptoms, (7) satisfaction with treatment, (8) side effects of treatment.These eight core outcomes reflect the joint priorities of postmenopausal women, clinicians, and researchers internationally. Standardized collection and reporting of these outcomes in clinical trials will facilitate the comparison of different treatments for genitourinary symptoms, advance clinical practice, and ultimately improve outcomes for symptomatic women.Video Summary:http://links.lww.com/MENO/A765 .

Abstract Aim The aim of the interdisciplinary S3-guideline Perimenopause and Postmenopause – Diagnosis and Interventions is to provide help to physicians as they inform women about the physiological changes which occur at this stage of life and the treatment options. The guideline should serve as a basis for decisions taken during routine medical care. This short version lists the statements and recommendations given in the long version of the guideline together with the evidence levels, the level of recommendation, and the strength of consensus. Methods The statements and recommendations are largely based on methodologically high-quality publications. The literature was evaluated by experts and mandate holders using evidence-based medicine (EbM) criteria. The search for evidence was carried out by the Essen Research Institute for Medical Management (EsFoMed). To some extent, this guideline also draws on an evaluation of the evidence used in the NICE guideline on Menopause and the S3-guidelines of the AWMF and has adapted parts of these guidelines. Recommendations Recommendations are given for the following subjects: diagnosis and therapeutic interventions for perimenopausal and postmenopausal women, urogynecology, cardiovascular disease, osteoporosis, dementia, depression, mood swings, hormone therapy and cancer risk, as well as primary ovarian insufficiency.

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.

Does resveratrol exert a potent inhibitory effect on the development of endometriosis by interfering with some pivotal processes?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.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.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.

Endothelin-1 (ET-1) is overexpressed in breast carcinomas and stimulates tumor cell growth in an autocrine and paracrine fashion via its receptors, ET(A)R and ET(B)R. In this study, we evaluated the expression of ET-1 and ET receptors in breast carcinomas and determined its clinical and prognostic significance.We analyzed expression of ET-1, ET(A)R, and ET(B)R in 176 breast carcinomas using a semiquantitative immunohistochemical approach. Statistical analysis of clinicopathological variables such as pT stage, pN stage, hormone receptor status, Her-2/neu amplification, histological grade, and long-term follow-up data were performed.We observed a moderate to strong cytoplasmic staining for ET-1 in 69 (43.1%), for ET(A)R in 74 (46.5%), and for ET(B)R in 86 (53.4%) cases of primary breast cancer. A correlation was found between increased ET-1 expression and its receptors with several clinicopathological parameters that characterize aggressive types of breast cancer, with the exception of increased ET(A)R and ET(B)R expression with positive estrogen receptor status. Elevated expression of ET-1, ET(A)R, and ET(B)R was more common in breast carcinomas of patients with lower disease-free survival time and overall survival. In addition, a statistically significant correlation was observed between ET(A)R expression and reduced disease-free survival time (P = 0.041). Interestingly, the prognostic impact of ET(A)R expression was shown to be more pronounced in the subgroup of patients with a putative favorable prognosis according to classic prognostic factors.Therefore, analysis of ET(A)R expression may improve the prediction of relapse and death and facilitate an individually based risk-directed adjuvant therapy in breast cancer patients.

Reduced activity of beta4-galactosyltransferase 7 (beta4GalT-7), an enzyme involved in synthesizing the glycosaminoglycan linkage region of proteoglycans, is associated with the progeroid form of Ehlers-Danlos syndrome (EDS). In the invertebrates Drosophila melanogaster and Caenorhabditis elegans, mutations in beta4GalT-7 affect biosynthesis of heparan sulfate (HS), a modulator of several biological processes relevant to wound repair. We have analyzed structural alterations of HS and their functional consequences in human beta4GalT-7 Arg270Cys mutant EDS and control fibroblasts. HS disaccharide analysis by reversed phase ion-pairing chromatography revealed a reduced sulfation degree of HS paralleled by altered immunostaining patterns for the phage-display anti-HS antibodies HS4E4 and RB4EA12 in beta4GalT-7 mutant fibroblasts. Real-time PCR-analysis of 44 genes involved in glycosaminoglycan biosynthesis indicated that the structural alterations in HS were not caused by differential regulation at the transcriptional level. Scratch wound closure was delayed in beta4GalT-7-deficient cells, which could be mimicked by enzymatic removal of HS in control cells. siRNA-mediated knockdown of beta4GalT-7 expression induced morphological changes in control fibroblasts which suggested altered cell-matrix interactions. Adhesion of beta4GalT-7 deficient cells to fibronectin was increased while actin stress fiber formation was impaired relative to control cells. Also collagen gel contraction was delayed in the beta4GalT-7 mutants which showed a reduced formation of pseudopodia and filopodia, less efficient penetration of the collagen gels and a diminished formation of collagen suprastructures. Our study suggests an HS-dependent basic mechanism behind the altered wound repair phenotype of beta4GalT-7-deficient EDS patients.

To study the function of syndecan-1 (SDC1) and its potential regulator miR-10b in endometriosis.Experimental laboratory study.University medical center.Not applicable.The human endometriotic cell line 12Z was transiently transfected with SDC1 small interfering RNA or miR-10b precursors and investigated for changes in cell behavior and gene expression. 12Z and primary eutopic endometrial stroma cells of two American Society for Reproductive Medicine class III endometriosis patients were transfected with miR-10b precursors to investigate posttranscriptional regulation of SDC1.Quantitative polymerase chain reaction, Western blotting, flow cytometry, 3' untranslated region luciferase assays, and zymography were used to measure miR-10b-dependent targeting of SDC1 and SDC1-dependent expression changes of proteases and interleukin-6. Altered cell behavior was monitored by Matrigel invasion assays, cell viability assays, and mitogen-activated protein kinase activation blots.Knockdown of SDC1 inhibited Matrigel invasiveness by >60% but did not affect cell viability. Interleukin-6 secretion, matrix metalloproteinase-9 expression, and matrix metalloproteinase-2 activity were reduced, whereas plasminogen activator inhibitor-1 protein expression was up-regulated. miR-10b overexpression significantly down-regulated SDC1, reduced Matrigel invasiveness by 20% and cell viability by 14%, and decreased mitogen-activated protein kinase activation in response to hepatocyte growth factor.Syndecan-1, a target of miR-10b, inhibits epithelial endometriotic cell invasiveness through down-regulation of metalloproteinase activity and interleukin-6.

Abstract Purpose While the stem cell marker Musashi-1 (MSI-1) has been identified as a key player in a wide array of malignancies, few findings exist on its prognostic relevance and relevance for cancer cell death and therapy resistance in breast cancer. Methods First, we determined prognostic relevance of MSI-1 in database analyses regarding multiple survival outcomes. To substantiate findings, MSI-1 was artificially downregulated in MCF-7 breast cancer cells and implications for cancer stem cell markers, cell apoptosis and apoptosis regulator p21, proliferation and radiation response were analyzed via flow cytometry and colony formation. Radiation-induced p21 expression changes were investigated using a dataset containing patient samples obtained before and after irradiation and own in vitro experiments. Results MSI-1 is a negative prognostic marker for disease-free and distant metastasis-free survival in breast cancer and tends to negatively influence overall survival. MSI-1 knockdown downregulated stem cell gene expression and proliferation, but increased p21 levels and apoptosis. Similar to the MSI-1 knockdown effect, p21 expression was strongly increased after irradiation and was expressed at even higher levels in MSI-1 knockdown cells after irradiation. Finally, combined use of MSI-1 silencing and irradiation reduced cancer cell survival. Conclusion MSI-1 is a prognostic marker in breast cancer. MSI-1 silencing downregulates proliferation while increasing apoptosis. The anti-proliferation mediator p21 was upregulated independently after both MSI-1 knockdown and irradiation and even more after both treatments combined, suggesting synergistic potential. Radio-sensitization effects after combining radiation and MSI-1 knockdown underline the potential of MSI-1 as a therapeutic target.

Triple-negative breast cancer (TNBC) is characterized by increased angiogenesis, metastasis, and poor survival. Dysregulation of the cell surface heparan sulfate proteoglycan and signaling co-receptor Syndecan-1 is linked to poor prognosis. To study its role in angiogenesis, we silenced Syndecan-1 in TNBC cell lines using a 3D human umbilical vein endothelial cell (HUVEC) co-culture system. Syndecan-1 siRNA depletion in SUM-149, MDA-MB-468, and MDA-MB-231 cells decreased HUVEC tubule network formation. Angiogenesis array revealed reduced VEGF-A and tissue factor (TF) in the Syndecan-1-silenced secretome. qPCR independently confirmed altered expression of F3, F7, F2R/PAR1, F2RL1/PAR2, VEGF-A, EDN1, IGFBP1, and IGFBP2 in SUM-149, MDA-MB-231, and MDA-MB-468 cells. ELISA revealed reduced secreted endothelin-1 (SUM-149, MDA-MB-468) and TF (all cell lines) upon Syndecan-1 depletion, while TF pathway inhibitor treatment impaired angiogenesis. Survival analysis of 3951 patients demonstrated that high expression of F3 and F7 are associated with better relapse-free survival, whereas poor survival was observed in TNBC and p53 mutant basal breast cancer (F3) and in ER-negative and HER2-positive breast cancer (F2R, F2RL1). STRING protein network analysis revealed associations of Syndecan-1 with VEGF-A and IGFBP1, further associated with the TF and ET-1 pathways. Our study suggests that TNBC Syndecan-1 regulates angiogenesis via the TF and additional angiogenic pathways and marks its constituents as novel prognostic markers and therapeutic targets.

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.

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.

<h2>Abstract</h2> Objective: To investigate microvessel density in adenomyosis compared to the endometrium in patients with adenomyosis and in normal controls. Design: Uterine paraffin-embedded histologic specimens were immunostained for CD34. The area with the highest microvessel density in adenomyosis and in the endometrium was evaluated. All microvessels in a specific field of view (×200 magnification) were counted. Setting: The Department of Gynecological Endocrinology and the Institute of Clinical Pathology, Department of Gynecopathology, in an university hospital. Patient(s): Specimens of 53 patients with adenomyosis, who had undergone hysterectomy. Endometrial specimens of 17 women without uterine pathology were investigated as normal controls. Main Outcome Measure(s): Microvessel density in adenomyosis and in the endometrium. Result(s): The mean microvessel density was significantly higher in adenomyosis than in the endometrium of the same patients (33.5 ± 14.6 vs. 19.5 ± 12.5 microvessels/field; <i>P</i><.001 sign test). No significant difference between the endometrium of patients and of normal controls was observed (<i>P</i>=.805). Conclusion(s): Adenomyosis exhibits angiogenic properties. However, the endometrium of patients with adenomyosis is not more prone to express angiogenic activity compared to the endometrium of normal controls.

Addition of gonadotropin releasing hormone to myo- [2-3H] inositol-prelabeled rat pituitary cells in primary culture evoked a dose-dependent increase of the accumulation of [3H] inositol phosphates with a rise of inositol triphosphate within 30 sec of stimulation, followed by a rise in inositol diphosphate and inositol monophosphate. Inositol phosphate accumulation was enhanced up to 5-to 8-fold and was time-dependent between up to 15 min incubation without further increase beyond this time period. Without preincubation with LiCl2, there was no measurable increase of GnRH-induced inositol phosphate accumulation compared to controls. The presence of calcium in the incubation medium did not affect the increase of inositol phosphates. These data give evidence, that polyphosphoinositide breakdown may be an early step in the action of gonadotropin releasing hormone on gonadotropin secretion.

To study whether insulin signaling pathways in the ovary are altered by metformin.In vitro human granulosa cell culture system.Academic research environment.Infertility patients undergoing oocyte retrieval for IVF/ICSI.Cell viability and phosphorylated protein kinase B (PKB/AKT) and p44/42 mitogen-activated protein kinase (MAPK) expression of human primary and HGL5 granulosa cells.Basal cell viability of primary granulosa cells was significantly increased relative to control by metformin preincubation, without an additional stimulatory effect of insulin or IGF. Phosphorylated AKT expression in lysates of the human granulosa cell line HGL5 was significantly increased in contrast to decreased phosphorylated MAPK expression by metformin preincubation.Besides systemic effects, the ovulation inducing action of metformin may at least partially be due to direct effects on insulin signaling intermediates and follicular growth patterns in the ovary.

Androgens and insulin are endocrine key players in the pathophysiology of polycystic ovary syndrome (PCOS), a heterogenic condition of unexplained etiology and a suspected genetic background. Androgens mediate the clinical phenotype of the disease. Therefore,all criteria of the recent PCOS consensus definition are based on their biological effects. Insulin resistance, followed by compensatory hyperinsulinemia, is frequently found in patients with PCOS. Insulin resistance is correlated with a risk of metabolic complications of PCOS, and recent research has focused on possible long-term health consequences of the syndrome. Newest molecular genetic findings at the receptor level of both androgens and insulin support their pivotal role in PCOS. These results could help to better characterize the heterogenic disorder, enabling a refinement of existing individualized therapeutic strategies.

Strain elastography (SE) is a new technique of parametric imaging that allows quantification of the elasticity of tissue. The aim of our study was to determine if the elasticity of para-urethral tissue correlates with urethral mobility and urinary incontinence (UI). Ninety-nine unselected women were investigated with SE. They were given a standardized interview about UI, and SE raw data for the para-urethral tissue were acquired in a sagittal standard urethra-symphysis view while being stimulated by a coughing fit. We placed one region of interest (ROI A) in the tissue between the urethra and vagina at midlevel of the urethra bordering the urethral wall. The second ROI (ROI B) was set at the level of the os urethra internum in the tissue of the bladder neck in one line to ROI A. We measured elasticity in both ROIs with TDI-Q (Tissue Doppler Imaging-Quantification Software) and calculated the ratio between ROI A and ROI B (A/B). Mobility of the urethra was quantified by measuring the angle between a line parallel to the urethra and a line parallel to the bladder neck during stress and rest. SE analysis was feasible in all cases. A/B was found to be correlated with the incidence of urethral mobility (p < 0.001). The incidence of UI was associated with an increase in urethral mobility (p = 0.04). No correlation between UI and A/B could be shown (p = 0.24). We observed a correlation between urethral mobility and elasticity of the para-urethral tissue. In case of increasing urethral mobility, the para-urethral tissue close to the bladder neck seems to be more elastic, and the patients reported about more symptoms of UI. No noticeable correlation between UI and urethral elasticity was shown. SE may be a useful technique for direct quantification of tissue elasticity and assessment of pelvic floor biomechanics.

Previous studies reported increased expression of the notch pathway-associated protein Musashi-1 in endometriosis. This case–control study investigates an association of the endometrial stem cell markers notch-1 and numb with endometriosis. Fifty-one endometriosis patients and 76 controls were recruited in the IVF unit and tertiary endometriosis referral centre of a university hospital. All subjects underwent transcervical endometrial biopsy and diagnostic laparoscopy. Expression of endometrial notch-1 and numb was assessed by immunostaining and correlated with clinical data. Association of stem-cell-marker expression with the presence of endometriosis was evaluated. Numb expression in the luminal epithelium was significantly higher in eutopic endometrium of endometriosis patients compared with controls (20.5% versus 16.5%, P = 0.033). Numb-positive single stromal cells were less frequent in endometrioma patients compared with other forms of endometriosis (0.3 versus 0.5 cells/visual field; P = 0.028). Notch-1 expression in endometrial glands was significantly higher in patients with deep infiltrating endometriosis compared with controls (39.1% versus 21.8%; P = 0.045). We conclude that stem cell markers notch-1 and numb of eutopic endometrium are associated with endometriosis and its clinical presentations, supporting the stem cell hypothesis of endometriosis. These findings could help develop promising research strategies applying endometrial stem cells as novel tools.

Heparan sulfate (HS) is a glycosaminoglycan found mainly in its protein-conjugated form at the cell surface and the extracellular matrix. Its high sulfation degree mediates functional interactions with positively charged amino acids in proteins. 2-O sulfation of iduronic acid and 3-O sulfation of glucosamine in HS are mediated by the sulfotransferases HS2ST and HS3ST, respectively, which are dysregulated in several cancers. Both sulfotransferases regulate breast cancer cell viability and invasion, but their role in cancer stem cells (CSCs) is unknown. Breast CSCs express characteristic markers such as CD44+/CD24-/low, CD133 and ALDH1 and are involved in tumor initiation, formation, and recurrence. We studied the influence of HS2ST1 and HS3ST2 overexpression on the CSC phenotype in breast cancer cell lines representative of the triple-negative (MDA-MB-231) and hormone-receptor positive subtype (MCF-7). The CD44+/CD24-/low phenotype was significantly reduced in MDA-MB-231 cells after overexpression of both enzymes, remaining unaltered in MCF-7 cells. ALDH1 activity was increased after HS2ST1 and HS3ST2 overexpression in MDA-MB-231 cells and reduced after HS2ST1 overexpression in MCF-7 cells. Colony and spheroid formation were increased after HS2ST1 and HS3ST2 overexpression in MCF-7 cells. Moreover, MDA-MB-231 cells overexpressing HS2ST1 formed more colonies and could not generate spheres. The phenotypic changes were associated with complex changes in the expression of the stemness-associated notch and Wnt-signaling pathways constituents, syndecans, heparanase and Sulf1. The results improve our understanding of BCSC function and mark a subtype-specific impact of HS modifications on the CSC phenotype of triple-negative and hormone receptor positive breast cancer model cell lines.

Syndecan-4, a predicted target of the microRNA miR-140-3p, plays an important role in multiple steps of tumor progression and is the second most abundant heparan sulfate proteoglycan produced by breast carcinoma cell lines. To investigate the potential functional relationship of miR-140-3p and syndecan-4, MDA-MB-231, SKBR3, and MCF-7 breast cancer (BC) cells were transiently transfected with pre-miR-140-3p, syndecan-4 small interfering RNAJ, or control reagents, respectively. Altered cell behavior was monitored by adhesion, migration, and invasion chamber assays. Moreover, the prognostic value of syndecan-4 was assessed by Kaplan-Maier Plotter analysis of gene expression data from tumor samples of 4929 patients. High expression of syndecan-4 was associated with better relapse-free survival in the whole collective of BC patients, but correlated with a worse survival in the subgroup of estrogen receptor negative and estrogen/progesterone-receptor negative patients. miR-140-3p expression was associated with improved survival irrespective of hormone receptor status. miR-140-3p overexpression induced posttranscriptional downregulation of syndecan-4, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and luciferase assays, resulting in decreased BC cell migration and matrigel invasiveness. Furthermore, miR-140-3p overexpression and syndecan-4 silencing increased the adhesion of BC to fibronectin and laminin. qPCR analysis demonstrated that syndecan-4 silencing leads to altered gene expression of adhesion-related molecules, such as fibronectin and focal adhesion kinase, as well as in the gene expression of the proinvasive factors matrix metalloproteinase 2 and heparanase (also known as HPSE). We conclude that syndecan-4 is a novel target of miR-140-3p that regulates BC cell invasiveness and cell-matrix interactions in the tumor microenvironment.

Abstract Breast cancer continues to be a serious public health problem. The role of the hedgehog pathway in normal development of the mammary gland as well as in carcinogenesis and progression of breast cancer is the subject of intense investigation, revealing functional interactions with cell surface heparan sulfate. Nevertheless, its influence on breast cancer prognosis, and its relation to specific sulfation motifs in heparan sulfate have only been poorly studied in large patient cohorts. Using the public database KMplotter that includes gene expression and survival data of 3951 patients, we found that the higher expression of SHH, HHAT, PTCH1, GLI1, GLI2 , and GLI3 positively influences breast cancer prognosis. Stratifying patients according to the expression of hormone receptors, histological grade, lymph node metastasis, and systemic therapy, we observed that GLI1 , GLI2 , and GLI3 expression, as well as co‐expression of SHH and ELP1 were associated with worse relapse‐free survival in patients with HER2‐positive tumors. Moreover, GLI1 expression in progesterone receptor‐negative tumors and GLI3 expression in grade 3 tumors correlated with poor prognosis. SHH , in a panel of cell lines representing different breast cancer subtypes, and HHAT, PTCH1, GLI1, GLI2 , and GLI3 were mostly expressed in cell lines classified as HER2‐positive and basal‐like. Expression of SHH, HHAT, GLI2 , and GLI3 was differentially affected by overexpression of the heparan sulfate sulfotransferases HS2ST1 and HS3ST2 in vitro. Although high HS2ST1 expression was associated with poor prognosis in KMplotter analysis, high levels of HS3ST2 were associated with a good prognosis, except for ER‐positive breast cancer. We suggest the GLI transcription factors as possible markers for the diagnosis, treatment, and prognosis of breast cancer especially in HER2‐positive tumors, but also in progesterone receptor‐negative and grade‐3 tumors. The pathway interaction and prognostic impact of specific heparan sulfate sulfotransferases provide novel perspectives regarding a therapeutical targeting of the hedgehog pathway in breast cancer.

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).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.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.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 objective of this study was to investigate expression of various growth factors associated with angiogenesis and lymphangiogenesis and of their receptors in ductal carcinomas in situ of the breast (DCIS). We studied protein expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF)-A, endothelin (ET)-1, and VEGF-C, and their receptors bFGF-R1, Flt-1, KDR, ET(A)R, ET(B)R, and Flt-4 immunohistochemically in 200 DCIS (pure DCIS: n=96; DCIS adjacent to an invasive component: n=104) using self-constructed tissue microarrays. Basic fibroblast growth factor-R1, VEGF-C, Flt-4, and ET(A)R were expressed in the tumour cells in the majority of cases, whereas bFGF and Flt-1 expression was rarely observed. VEGF-A, KDR, ET-1, and ET(B)R were variably expressed. The findings of VEGF-C and its receptor Flt-4 as lymphangiogenic factors being expressed in tumour cells of nearly all DCIS lesions and the observed expression of various angiogenic growth factors in most DCIS suggest that in situ carcinomas are capable of inducing angiogenesis and lymphangiogenesis. Moreover, we found a higher angiogenic activity in pure DCIS as compared to DCIS with concomitant invasive carcinoma. This association of angiogenic factors with pure DCIS was considerably more pronounced in the subgroup of non-high-grade DCIS (n=103) as compared with high-grade DCIS (n=94). Determination of these angiogenic markers may therefore facilitate discrimination between biologically different subgroups of DCIS and could help to identify a particularly angiogenic subset with a potentially higher probability of recurrence or of progression to invasiveness. For these DCIS, targeting angiogenesis may represent a feasible therapeutic approach for prevention of progression of DCIS to invasion.

This study was undertaken to evaluate the feasibility of autofluorescence laparoscopy in the diagnosis of endometriotic lesions.Prospective analysis of 83 consecutive patients undergoing laparoscopy for suspected endometriosis under white light illumination and autofluorescence diagnosis. The study measured total number of endometriotic lesions diagnosed under white light illumination and with autofluorescence diagnosis.The biopsy-based sensitivity of white light diagnosis alone and white light illumination and autofluorescence for detecting nonpigmented peritoneal endometriotic lesions was 65% compared with 92% (1.42-fold increase). The corresponding specificity was 68% as opposed to 84%. Occult areas of endometriosis were discovered using autofluorescence diagnosis. Statistical analysis was performed with chi2 test and McNemar test.Combination of white light illumination and autofluorescence is significantly superior to white light illumination alone in detecting nonpigmented endometriotic lesions. Autofluorescence diagnosis of nonpigmented endometriotic lesions may become an alternative to fluorescence diagnosis after application of 5-aminolevulinic acid, especially because of no side effects.

Triple-negative breast cancer (TNBC) is defined by a lack of hormone receptor expression as well as lack of overexpression/amplification of HER2/neu. Patients with TNBC show a significantly worse prognosis compared to patients with other breast cancer subtypes. TNBC, however, is a heterogeneous entity both with regard to clinical/pathological characteristics and molecular biology. This review summarizes the current data on TNBC with a particular focus on mutational and gene expression profiling and the association between TNBC and breast cancer stem cells. Das tripelnegative Mammakarzinom (triple negative breast cancer, TNBC) ist definiert durch eine fehlende Hormonrezeptorexpression sowie eine fehlende Amplifikation/Überexpression des HER2/neu-Onkogens. Patientinnen mit einem TNBC zeigen eine signifikant schlechtere Prognose im Vergleich zu Patientinnen mit anderen Mammakarzinomsubtypen. Jedoch handelt es sich beim TNBC durchaus um eine heterogene Erkrankung sowohl im Hinblick auf klinische/pathologische Parameter als auch auf molekularbiologische Faktoren. In dieser Übersichtsarbeit fassen wir die aktuellen Daten zum TNBC zusammen mit einem speziellen Fokus einerseits auf die Ergebnisse von Mutations- und Genexpressionsanalysen, andererseits auf den Zusammenhang zwischen TNBC und Mammakarzinom-Stammzellen.

Background: Triple negative breast cancer (TNBC) is characterized by lack of expression of both estrogen and progesterone receptor as well as lack of amplification of HER2. Patients with TNBC carry an unfavorable prognosis compared to other breast cancer subtypes given that endocrine or HER2 targeted therapies are not effective, rendering chemotherapy the sole effective treatment option to date. Therefore, there is a high demand for additional novel treatment options.Findings: We previously published a list of genes showing both higher gene expression rates in TNBC and, in addition, are known to encode targets of non-oncologic drugs. SRD5A1, which encodes the type-1 isoform of the steroid-5alpha-reductase, which is involved in androgen metabolism, was found to be one of these genes. Dutasteride is a dual blocker of both the type-1 and type-2 isoform of SRD5A1 and is indicated in the treatment of benign prostate hyperplasia. Treatment of TNBC cell lines with dutasteride was associated with a dose-dependent decrease in cell viability, altered protein expression of VEGF and HIF-1α and increased chemosensitivity.Conclusion: Our results demonstrate that the SRD5A1-corresponding anti-androgenic drug dutasteride might act as a combinatorial therapeutic option besides standard chemotherapy in highly aggressive TNBC.

Endometriosis is associated with severe pelvic pain and reduced fertility. Recently, it has been linked to a dysregulation of microRNAs (miRNAs), which are post-transcriptional regulators of gene expression. The functional effect of dysregulated miR-142-3p expression in endometrial stroma cells was investigated. An increased expression of miR-142-3p resulted in a significantly reduced expression of steroid sulfatase and interleukin-6-coreceptor gp130 as well as reduced interleukin-6-mediated activation of the STAT3-pathway, suggesting an effect of miR-142-3p both on steroid hormone- and cytokine-mediated signalling events. At the functional level, miR-142-3p overexpression significantly reduced cell viability (P ≤ 0.01). miR-142-3p regulation emerges as a future therapeutic strategy for endometriosis.