Monika M. Wölfler

Hematopoietic Stem and Progenitor Cells Acquire Distinct DNA-Hypermethylation During in vitro Culture

Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses stemness during culture. In this study, we compared DNA-methylation (DNAm) profiles of freshly isolated and culture-expanded HPCs. Culture conditions of CD34+ cells - either with or without mesenchymal stromal cells (MSCs) - had relatively little impact on DNAm, although proliferation is greatly increased by stromal support. However, all cultured HPCs - even those which remained CD34+ - acquired significant DNA-hypermethylation. DNA-hypermethylation occurred particularly in up-stream promoter regions, shore-regions of CpG islands, binding sites for PU.1, HOXA5 and RUNX1, and it was reflected in differential gene expression and variant transcripts of DNMT3A. Low concentrations of DNAm inhibitors slightly increased the frequency of colony-forming unit initiating cells. Our results demonstrate that HPCs acquire DNA-hypermethylation at specific sites in the genome which is relevant for the rapid loss of stemness during in vitro manipulation.

Two-dimensional gel electrophoresis in peritoneal fluid samples identifies differential protein regulation in patients suffering from peritoneal or ovarian endometriosis

Endometriosis is determined by local and systemic proinflammatory dysregulation and therefore differential protein expression in peritoneal fluid (PF). Of highest interest is lesion formation and the establishment and persistence of endometriosis. In this study we analyzed well-characterized PF samples of patients with ovarian or peritoneal endometriosis and compared them to control samples. We found 11 proteins differentially regulated, of which some might play a key role in the pathogenesis of endometriosis.

Reduced hemopexin levels in peritoneal fluid of patients with endometriosis

OBJECTIVE To study altered hemopexin concentrations in peritoneal fluid (PF) samples from patients with endometriosis. Recent data implicate a role of altered iron metabolism in endometriosis patients. Hemopexin is the major transport protein for heme. Like iron, heme exposure to the epithelial surface can provoke oxidative stress on the peritoneal epithelium. Therefore, altered hemopexin concentrations and heme scavenging in PF might play a role in the pathophysiology of endometriosis. DESIGN Prospective explorative study. SETTING Academic tertiary care center. PATIENT(S) Eighty symptomatic patients scheduled for laparoscopy for the diagnosis and/or therapy of endometriosis. INTERVENTION(S) Aspiration of PF samples during laparoscopy. MAIN OUTCOME MEASURE(S) Hemopexin and heme concentration in PF. RESULT(S) At laparoscopy, 47 of 80 (58.8%) patients exhibited endometriosis, and 33 (41.2%) were proven disease-free (CO). By means of ELISA significantly lower concentrations of hemopexin in the samples from patients with endometriosis (endometriosis 0.377 ± 0.16 mg/mL) compared with controls (disease-free 0.479 ± 0.20 mg/mL) could be demonstrated. Heme levels in the samples were not significantly different between groups (endometriosis 9.130 ± 6.124 μM and disease-free 9.990 ± 4.485 μM). There was no significant correlation between heme and hemopexin levels (Pearsons correlation coefficient r = -0.146). Demographic data between the groups were comparable. CONCLUSION(S) These data provide further evidence that hemopexin is significantly down-regulated in PF samples from patients with endometriosis compared with controls. This study confirms recent findings in two-dimensional gel electrophoresis demonstrating a down-regulation of hemopexin in PF from patients with endometriosis in a larger series of samples.

Laparoskopie in der gynäkologischen Onkologie

ZusammenfassungHintergrundGynäkologische Malignome werden zunehmend laparoskopisch operiert.Ziel der ArbeitDer vorliegende Beitrag gibt Antworten zu folgenden Fragen: Bei welcher Indikation in der gynäkologischen Onkologie kann laparoskopisch operiert werden? Ist eine der offenen Chirurgie vergleichbare Sicherheit gegeben?Material und MethodeEs erfolgte eine Auswertung der Literatur unter besonderer Berücksichtigung prospektiver randomisierter Studien. Der vorliegende Beitrag fasst die wesentlichen Publikationen endoskopischer Operationsverfahren beim Endometrium-, Zervix- und Ovarialkarzinom sowie der Borderline-Tumoren des Ovars zusammen.ErgebnisseDie operative Therapie von Frühstadien des Endometrium- und Zervixkarzinoms, einschließlich der pelvinen und paraaortalen Lymphonodektomie, kann laparoskopisch durchgeführt werden. Die bisherige Datenlage lässt auf eine der Laparotomie vergleichbare onkologische Sicherheit schließen. Für das Zervixkarzinom wird beim lokal fortgeschrittenen Tumor ein endoskopisches operatives Staging zur Bestimmung einer intraperitonealen Ausbreitung und des paraaortalen Lymphknotenstatus zunehmend häufiger durchgeführt. Für Frühstadien des Ovarialkarzinoms wird aufgrund einer umfassenderen Evaluationsmöglichkeit die offene Operation empfohlen, zumal keine randomisierte Studie zum Vergleich von Laparoskopie und Laparotomie existiert. Einen wichtigen Stellenwert hat die Laparoskopie zur Diagnostik bei unklaren ovariellen Befunden sowie bei der Fokussuche im Fall eines „Cancer-of-unknown-primary“(CUP)-Syndroms und zur Gewebeentnahme vor einer neoadjuvanten Therapie. Einen festen Platz besitzt die Laparoskopie bei der Operation ovarieller Borderline-Tumoren.SchlussfolgerungenFrühstadien des Endometrium- und des Zervixkarzinoms sowie Borderline-Tumoren des Ovars können laparoskopisch sicher operiert werden. Alle invasiven Ovarialkarzinome sollen offen operiert werden. Bei fortgeschrittenen Stadien gynäkologischer Malignome wird die Laparoskopie zur Diagnostik eingesetzt, insbesondere beim Zervixkarzinom zur Evaluation des paraortalen Lymphknotenstatus und beim Ovarialkarzinom zur Gewebeentnahme vor einer geplanten neoadjuvanten Therapie.AbstractBackgroundLaparoscopy is increasingly being used for operative treatment of gynecological malignancies.AimThis article supplies answers to the following questions: what are the indications in gynecological oncology for laparoscopic operations? Does one of the open surgical procedures offer comparable oncological safety?Material and methodsAn analysis of the literature was carried out with respect to prospective randomized trials. This article summarizes the important publications on endoscopic operative procedures for endometrial, cervical and ovarian cancer as well as borderline ovarian tumors.ResultsOperative treatment of early stage endometrial and cervical cancer can be carried out using laparoscopy, including pelvic and para-aortic lymphadenectomy. The current data suggest that there is comparable oncological safety between laparoscopy and laparotomy. Surgical staging can be used to assess the retroperitoneal para-aortic lymph node status before chemoradiation of locally advanced cervical cancer. Operative treatment of early stage ovarian cancer should be carried out via midline incision laparotomy because of a superior evaluation of the bowels, retroperitoneum and upper abdomen. Laparoscopy plays an important role in the diagnostics of cancer of unknown primary (CUP) syndrome and to acquire tissue biopsies before neoadjuvant chemotherapy. Borderline ovarian tumors can be treated laparoscopically without impairing patient prognosis.ConclusionsLaparoscopy of early stage endometrial and cervical cancer and borderline ovarian tumors can be performed with oncological safety and comparable prognosis to open surgery. Invasive ovarian cancer should be treated via open surgery. In cases of advanced stage gynecological malignancies laparoscopy provides a diagnostic tool facilitating the assessment of the para-aortic lymph node status before chemoradiation of cervical cancer and allows acquisition of tissue biopsies of ovarian cancer before neoadjuvant chemotherapy.

Fatigue – a symptom in endometriosis

STUDY QUESTION Is fatigue a frequent symptom of endometriosis? SUMMARY ANSWER Fatigue is an underestimated symptom of endometriosis as it affects the majority of women with endometriosis, but it is not widely discussed in literature. WHAT IS KNOWN ALREADY Fatigue can be a symptom of endometriosis causing major distress impacting the daily activities and quality of life of women with endometriosis. However, few studies with large sample sizes have investigated fatigue as a symptom of endometriosis. STUDY DESIGN, SIZE, DURATION The study was designed as a multi-center matched case-control study. Recruitment took place at hospitals and private practices in Switzerland, Germany and Austria between 2010 and 2016. Data was collected from 1120 women, 560 of them with endometriosis. The women with endometriosis were matched to 560 control women in regard to age ±3 years and ethnic background. PARTICIPANTS/MATERIALS, SETTING, METHODS Diagnosis of women with endometriosis had to be surgically and histologically confirmed. Surgical exclusion or absence of any endometriosis-identifying symptoms was required for control subjects. Materials included surgical and histological reports as well as data retrieved from a self-administered questionnaire. This study focused on the symptom fatigue in endometriosis. Relationships of variables were established by regression analysis and associations were quantified as odds ratios. MAIN RESULTS AND THE ROLE OF CHANCE Frequent fatigue was experienced by a majority of women diagnosed with endometriosis (50.7% versus 22.4% in control women, P < 0.001). Fatigue in endometriosis was associated with insomnia (OR: 7.31, CI: 4.62-11.56, P < 0.001), depression (OR: 4.45, CI: 2.76-7.19, P < 0.001), pain (OR: 2.22, CI: 1.52-3.23, P < 0.001), and occupational stress (OR: 1.45, CI: 1.02-2.07, P = 0.037), but was independent of age, time since first diagnosis and stage of the disease. LIMITATIONS, REASONS FOR CAUTION Women with asymptomatic endometriosis cannot be excluded in the control group which would lead to underestimation of our results. The studys design allows no evaluation of causal effects. WIDER IMPLICATIONS OF THE FINDINGS As fatigue is experienced by numerous women with endometriosis, it needs to be addressed in the discussion of management and treatment of the disease. In addition to treating endometriosis, it would be beneficial to reduce insomnia, depression, pain and occupational stress in order to better manage fatigue. STUDY FUNDING/COMPETING INTEREST(s) There was no additional funding received for this study and no conflict of interest. TRIAL REGISTRATION NUMBER ClinicalTrials.gov, NCT 02511626.

Hysterektomie: interventionelle Alternativen

ZusammenfassungIst eine Gebärmutterentfernung aus medizinischen Gründen indiziert oder besteht der Wunsch nach sicherer Beschwerdefreiheit, werden zur Vermeidung einer abdominellen Hysterektomie (HE) heutzutage bevorzugt die vaginale HE sowie laparoskopisch assistierte Verfahren, wie die totale laparoskopische Hysterektomie (TLH), die laparoskopisch assistierte suprazervikale Hysterektomie (LASH) und die laparoskopisch assistierte vaginale Hysterektomie (LAVH), angewandt. Die Art des Verfahrens richtet sich nach der Diagnose, dem Wunsch der Patientin sowie der fachlichen Expertise. Eine HE ist in einigen Fällen nicht indiziert und kann vor allem bei Myomen und Blutungsstörungen häufig durch alternative, organerhaltende Verfahren ersetzt werden. Myomenukleationen sowie neuere Verfahren der Myomembolisation und der MRT-gesteuerten fokussierten Sonographie stehen dabei im Vordergrund. Eine Verödung der Gebärmutterschleimhaut bei Blutungsstörungen kann nach Ausschluss eines Malignoms und nach abgeschlossenem Kinderwunsch durch eine Endometriumablation zur Verbesserung bzw. zum Sistieren der Beschwerden führen. Zunehmend wird auch die Roboterchirugie zur Hysterektomie und Myomenukleation angewandt.AbstractIf hysterectomy is medically indicated or there is a patient wish for guaranteed freedom from suffering, nowadays in order to avoid an abdominal hysterectomy, the preferred approaches are vaginal hysterectomy as well as laparoscopically-assisted procedures, such as total laparoscopic hysterectomy (TLH), laparoscopically-assisted supracervical hysterectomy (LASH) and laparoscopically-assisted vaginal hysterectomy (LAVH). The type of procedure used depends on the diagnosis, the wishes of the patient and the technical competence. Hysterectomy is not indicated in some cases and can often be replaced by organ preserving procedures especially for myomas and bleeding disorders. Enucleation of myomas and new procedures of myoembolization and magnetic resonance imaging-guided focused sonography are of great importance. An atrophy of the cervical mucosa in bleeding disorders by endometrial ablation can lead to an improvement and reduction of symptoms after exclusion of a malignancy and where there is no desire to have children. Robot-assisted surgery is being used increasingly more for hysterectomy and myoma enucleation.

Neue Aspekte zur Ätiologie und Pathogenese der Endometriose

ZusammenfassungHintergrundEndometriose stellt durch die vielfältige Morphologie und diverse Beschwerdebilder eine schwierig fassbare Entität dar. Ihre Entstehung ist noch teilweise ungeklärt. Stand der ForschungFest steht, dass uterine Dysperistalsis eine verstärkte retrograde Menstruation nach sich zieht, was für Oberflächenschädigung am Peritoneum prädisponiert und so wohl die Implantation disseminierter endometrialer Zellen erleichtert wird. Das eutope Endometrium von Frauen mit Endometriose zeigt viele Veränderungen im Vergleich zu Kontrollen, die ektopen endometrialen Zellen der Endometrioseläsionen selbst haben ein verändertes Potenzial für Adhäsion, Invasion und Proliferation. Neben der Fähigkeit zur lokalen Östrogensynthese und relativen Progesteronresistenz weisen rezente Studien auf die Bedeutung epigenetischer Veränderungen hin. Durch die immunologische Dysfunktion bei Endometriose besteht einerseits eine verminderte lokale Immunreaktion mit reduzierter peritonealer Clearance, welche die Implantation von endometrialen Zellen nach retrograder Menstruation erleichtert, andererseits unterstützt ein proinflammatorisches Mikromilieu die Etablierung von Endometrioseläsionen; beide Mechanismen sind für die Ätiologie und Pathogenese zentral. AusblickRezente Studien deuten zunehmend auf eine Beteiligung von endometrialen Stammzellen hin. Aufgrund ihres hohen proliferativen Potenzials, erweiterter Differenzierungsmöglichkeiten und der enormen Regenerationsfähigkeit begünstigen endometriale Stammzellen die Entstehung von Endometriose.AbstractBackgroundEndometriosis is one of the most common benign diseases in the field of gynecology; however, after years of extensive research the pathogenesis remains enigmatic. State of researchIt is known that increased retrograde menstruation caused by uterine dysperistalsis and hyperpersistalsis and subsequent increased oxidative stress on the peritoneal mesothelium are vital factors facilitating the implantation of disseminated endometrial cells and endometriotic lesion formation. The eutopic endometrium in endometriosis patients appears to have a differential protein expression when compared to controls and ectopic endometrial cells of endometriotic lesions exhibit an altered potential for adhesion, invasion and proliferation. The capability for local estrogen biosynthesis and relative progesterone resistance as well as recently demonstrated epigenetic alterations of endometrial cells are key features for the development of this disease. Endometriosis is associated with an immunological dysfunction. Impaired immunosurveillance and peritoneal clearance on the one hand and the proinflammatory microenvironment on the other hand are considered to be key factors for the development of endometriotic lesions. PerspectivesRecent data indicate the relevance of endometrial stem cells in the etiology and pathogenesis of endometriosis. Due to the high proliferative potential, extended cell differentiation capacity and tremendous ability for regeneration, endometrial stem cells are in the focus of research activities.

Proteomische Techniken zur Diagnostik der Endometriose

ZusammenfassungDie Endometriose ist eine der häufigsten Erkrankungen bei Frauen im Reproduktionsalter und für die Diagnostik eine schwer zu fassende Entität. Durch einfache Proteinbestimmungen und proteomische Techniken wie Massenspektrometrie (MS) und 2-D-Gelelektrophorese (2-DE) können pathognomonische Prozesse bei der Endometriose im Vergleich zur physiologischen Situation untersucht werden. Bekannte bei der Endometriose differenziell exprimierte Proteine können bereits einzeln oder in Biomarkerpanels als diagnostische Marker eingesetzt werden. Umfangreichere Untersuchungstechniken wie die „protein pattern profiling MS“, die mit hoher Sensitivität und Spezifität die Erkrankung vorhersagen können, stellen dagegen aufgrund der hohen Kosten eher experimentelle Ansätze dar.Die 2-DE ist eine hervorragende Methode zur Untersuchung bisher unbekannter Regulationsmechanismen bei der Endometriose. Durch die nachfolgende MS-Analyse zur Proteinidentifizierung der differenziell regulierten Proteine können neue diagnostische und möglicherweise auch therapeutische Ziele identifiziert werden.AbstractEndometriosis is one of the most common benign diseases among premenopausal women. However, after years of extensive research, it is still impossible to correctly diagnose endometriosis without an operation. Protein detection and proteomic techniques like two-dimensional gelelectrophoresis (2DE) and mass spectrometry (MS) are applicable to explore pathognomonic processes by comparison of sera from endometriosis patients and from normal controls, respectively. Therefore, these techniques may reveal novel diagnostic markers for endometriosis. While single protein markers or panels of markers may presently enter clinical application, “high throughput” screening techniques like protein pattern profiling remain experimental due to exalted technology and costs. Moreover, 2DE is an excellent technique to research unknown pathways and detect differentially expressed proteins when additionally subjected to tandem mass spectrometry (MS/MS) for protein identification. This might lead to the discovery of new diagnostic and potentially therapeutic markers.

Proteomics translational@@@Translational proteomics: Praxis oder Theorie?@@@Practice or theory?

ZusammenfassungDas Proteom bietet in seiner Komplexität ein präzises Abbild der zellulären Vitalität und Funktion. Proteomische Techniken, wie 2D-Gelelektrophorese (2DE) und Massenspektrometrie (MS), bieten vielfältige Möglichkeiten, pathognomonische Prozesse zu untersuchen und im Vergleich zwischen normalen und veränderten Entitäten differenziell exprimierte Proteine darzustellen. Damit können einerseits wertvolle Erkenntnisse über Fehlregulationen im Krankheitsfall gewonnen werden, andererseits liefern proteomische Untersuchungen diagnostisch verwertbare Informationen, die als Grundlage zur Früherkennung von Fehlregulationen und Erkrankungsneigung dienen können. Mittels 2DE können einzelne differenziell exprimierte Proteine dargestellt und durch spezielle MS-Verfahren (Tandem-MS, MS-MS) identifiziert werden. Die MS kann auch im direkten Ansatz zur „High-throughput“-Analyse von Proben mittels komplexer Mustererkennung („protein pattern profiling“) eingesetzt werden. Zahlreiche wissenschaftliche Publikationen und erste klinische Anwendungsversuche erscheinen erfolgversprechend.AbstractThe proteome is a complex but precise reflection of cellular vitality and function. Proteomic techniques such as 2D gel electrophoresis (2DE) and mass spectrometry (MS) are employed to investigate pathognomonic processes and discover differentially expressed proteins when comparing normal and diseased entities. On the one hand proteomic research provides valuable findings on dysregulations in disease, while on the other it provides diagnostically relevant information which could form the basis for early detection of dysregulation and disease predisposition. 2DE is an excellent technique to detect differentially expressed proteins which are subjected to tandem mass spectrometry (MS-MS) for protein identification. Moreover, MS is applied for complex protein pattern profiling to analyze samples by means of “high throughput” screening and allocation to specific pathognomonic alterations. A series of scientific publications reporting on initial clinical applications appear promising in the search of prediction markers.