Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions.

BACKGROUND: A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses. METHODS: EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2 × 107 cells were stained with AlexaFluor 488-conjugated specific antibodies or corresponding isotype controls. Amount and size of particles in normal scattering light mode (N mode) versus fluorescence mode (F mode, laser wavelength 488 nm) was measured using ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Cryo electron microscopy (EM) was used to verify the presence of membrane bilayer surrounded nanoparticles. RESULTS: All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit. CONCLUSION: Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.

Red blood cell alloimmunization in 184 patients with myeloid neoplasms treated with azacitidine - A retrospective single center experience.

Alloimmunization to Red Blood Cell (RBC) antigens frequently occurs in patients with myeloid neoplasms (AML, MDS and CMML) and potentially poses the patient at risk for delayed hemolytic transfusion reactions and limited supply of compatible RBC-units. However, there is comparatively little data on transfusion associated characteristics in this patient cohort. We therefore retrospectively analyzed transfusion requirements and clinical outcomes of 184 patients with myloid neoplasms treated with azacitidine at the Paracelsus Medical University Salzburg, which were included in the Austrian Registry of Hypomethylating Agents. The mean blood component requirements for AML, MDS and CMML were 39.8, 67.4 and 31.4 RBC units and 31.7, 27.6 and 19.1 platelet (PLT) units respectively. In spite of an extended and stringent RBC unit matching policy (ABO, RhD, RhCcEe and K antigens), 20 (11%) patients formed at least one alloantibody ("allo-group"), whereas 164 patients (89%) did not ("non-allo-group"). The most frequent antibody specificity was anti-E, followed by anti-Wra -Lua, -D, -C and -Jka. Alloimmunization was associated with higher numbers of transfused RBC units (68 vs. 38; p=0.001), as well as with longer time under transfusion (16.7 vs. 9.4 months; p=0.014). Median overall survival (OS) did not differ significantly between the "allo"- and "non-allo-group".

T-cell death, phosphatidylserine exposure and reduced proliferation rate to validate extracorporeal photochemotherapy.

BACKGROUND AND OBJECTIVES: Extracorporeal photochemotherapy (ECP) is an established therapy in various diseases, such as cutaneous T-cell lymphoma and graft-versus-host disease. This study was performed to investigate the practicability of a flow cytometric T-cell evaluation after ECP as a tool to validate the quality of ECP procedures and to enable the comparability of treatments with different ECP devices. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMNCs) of healthy volunteer blood donors were treated by offline ECP. To quantify the effect of ECP on T cells in vitro, phosphatidylserine exposure and 7-aminoactinomycin D (7-AAD) reactivity as well as the proliferative activity of phytohaemagglutinin-induced, viable CD3(+) lymphocytes were analysed by flow cytometry. RESULTS: The expected T-cell death after ECP was confirmed by 7-AAD measurements. Phosphatidylserine exposure gradually increased between 20 and 70 h after ECP. Treatment-related inhibition of T-cell proliferation was 92.6 ± 1.4%. CONCLUSION: The combination of viability, phosphatidylserine exposure and T-cell division analyses by flow cytometry in a single-platform system provides a valuable tool to validate ECP procedures.

Reticulocyte hemoglobin content allows early and reliable detection of functional iron deficiency in blood donors.

BACKGROUND: Frequent blood donations may lead to a depletion of body iron stores resulting in manifest anemia. Reticulocyte hemoglobin content (CHr) - a marker for impaired hemoglobinisation (IH) caused by functional iron deficiency (FID) - was investigated regarding its value as a routine screening parameter in frequent whole blood donors. METHODS: In a prospective study, 917 frequent blood donors and 688 first time or reactivated donors were tested for iron status and red blood cell count, including CHr. The ferritin index as a marker to indicate absent iron stores (AIS) was calculated. RESULTS: Depending on the number of donations during the preceding 12 months, AIS were detected in up to 21.4% of male and 27.8% of female donors, respectively. IH was present in up to 6.4% male and 16.7% female donors with 2 and 4 preceding donations, respectively. The defined CHr cut-off value was 28.0 pg to detect IH in frequent whole blood donors with AIS, leading to a test specificity of 98.2% (positive predictive value, PPV: 57.7%) in male and of 97.8% (PPV: 82.9%) in female donors. CONCLUSION: Determination of CHr is feasible to detect FID resulting in IH in frequent blood donors. It may help to prevent the development of anemia in frequent blood donors and also can help to decide whether donor deferral or even iron substitution need to be recommended.

Function and activation state of platelets in vitro depend on apheresis modality.

BACKGROUND AND OBJECTIVES: In multicomponent collection, various blood components are prepared during one apheresis process. The aim of this prospective crossover study was to compare the function, metabolic parameters and activation state of fresh and stored platelets (PLTs) collected by two different cell separators. MATERIALS AND METHODS: Twenty-four donors underwent apheresis on each of two cell separators (Fenwal Amicus(®) and CaridianBCT Trima Accel(®)) with an interval of at least 2 months between donations. Per donation, one double dose of PLT concentrate (PC) and one unit of packed red-blood-cells were collected. In total, 48 single unit PCs were tested for pH, glucose, bicarbonate, lactate, potassium and LDH concentration during 7 days of storage. PLT function was analysed by aggregometry, rotation thrombelastometry and hypotonic shock response. The PLT surface expression of P-selectin (CD62P) and LAMP-3 (CD63) was estimated by flow cytometry. RESULTS: During storage, metabolic parameters were well maintained in both groups, but levels of glucose and pH were significantly lower, while lactate and LDH were significantly higher in Amicus(®)-PCs. Amicus(®)-derived PLTs were significantly more activated as evidenced by higher CD62P and CD63 expression. In parallel, the in vitro function of Amicus(®)-PLTs was significantly reduced compared to Trima(®)-PLTs. CONCLUSION: In multicomponent apheresis, standardized PLT collection is effective and well tolerated. The higher activation of Amicus(®)-derived PLTs may be because of the divergent centrifugation modalities during collection. Possible consequences for the clinical outcome of thrombocytopenic patients will be evaluated in further trials.

Effect of skin surface temperature on skin pigmentation during contact and intralesional cryosurgery of keloids.

BACKGROUND: This 15-month study was designed to compare the effect of skin surface temperature on skin pigmentation following a single intralesional or contact cryosurgical treatment of keloids. PATIENTS/METHODS: Thirty Caucasian patients with 45 keloids present for more than 6 months were included in this study. Twenty-one keloids were treated by the contact method while the remaining 24 scars were managed using an intralesional cryosurgery technique. The skin surface temperature at the keloids was measured and recorded using a Ni/Cd thermocouple. Four variables of the thermal history were evaluated with the contact and the intralesional methods, namely cooling rate, hold time, end temperature and thawing rate. Assessment of the local hypopigmentation was performed 6 months after the treatment using a pigmentation scale. RESULTS: Significantly slower cooling (6.09 +/- 4.56 degrees C/min) and thawing rates (54.52 +/- 32.17 degrees C/min) were recorded with the intralesional cryosurgery method when compared with the cooling rates (13.47 +/- 9.04 degrees C/min) and thawing rates (89.00 +/- 86.42 degrees C/min) of the contact method (P < 0.000001). The end temperature of the contact technique was significantly cooler (-46.77 +/- 14.74 degrees C) when compared with that of the intralesional method (-15.55 +/- 6.77 degrees C) (P < 0.000001). There was a trend for the hold time of intralesional cryosurgery to be longer (82.67 +/- 138.03 s) than that of the contact method (16.86 +/- 23.49 s) (P < 0.059). A significant difference in skin pigmentation was demonstrated between the two cryosurgical methods. In 91.7% of the keloids treated by the contact technique a significant hypopigmentation was noticed, while no marked hypopigmentation was detected in the skin surface of the keloids treated by the intralesional method (P < 0.0001). CONCLUSION: We hypothesize that the thermal history of the skin surface during the intralesional cryosurgery technique provides a better survival environment for the melanocytes than the contact method, thus producing a lower rate of permanent hypopigmentation and disfiguring.

[Investigation of adhesion molecules (sVCAM-1) in serum of nonpregnant women, normotensive pregnant women and in patients with pregnancy complications]. / Nachweis von Adhäsionsmolekülen (sVCAM-1) im Serum von Nichtschwangeren, normotensiven Schwangeren sowie bei Patientinnen mit Erkrankungen während der Schwangerschaft.

OBJECTIVE: An increased expression of endothelial adhesion molecules combined with neutrophil activation in the placental bed is to be assumed aetiopathogenetically relevant in preeclampsia. MATERIAL AND METHODS: Ranges of sVCAM-1 serum concentrations of both control persons (29 nonpregnant and 25 normotensive pregnant women) and patients with different complications of pregnancy (HELLP-syndrome n = 10, preeclampsia n = 12, gestational hypertension n = 38, diabetes n = 24, growth retardation n = 21) were determined by means of ELISA. Frozen placental samples of 5 normotensive and 10 hypertensive pregnant women were investigated immunhistochemically to study the distribution of VCAM-1 in the placenta. RESULTS: Significantly elevated sVCAM-1 serum levels (p < 0.05) were identified in samples of patients with HELLP syndrome, preeclampsia, diabetes and gestational hypertension compared with serum levels of normotensive pregnant women. The cut-off level (97.5% percentile of normotensive serum levels) was calculated (775 ng/ml). VCAM-1 was localized immunhistochemically at capillaries of villi and main villi. CONCLUSIONS: There are closed relations between elevated serum levels of sVCAM-1 during pregnancy and diseases with vasculopathies of placental bed.

Megakaryocyte hyperplasia and enhanced agonist-induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice.

Vasodilator-stimulated phosphoprotein (VASP), a substrate of cAMP- and cGMP-dependent protein kinases, is associated with focal adhesions, cell-cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and the Listeria monocytogenes surface protein ActA. VASP is a member of the enabled (Ena)/VASP protein family and is thought to be involved in actin filament formation and integrin alphaIIbbeta3 inhibition in human platelets. To gain further insight into the in vivo function of this protein, VASP-deficient mice were generated by homologous recombination. VASP-/- mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin alphaIIbbeta3 activation. Although the limited phenotypic differences between wild-type and VASP-/- mice suggested functional compensation of VASP by members of the Ena/VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.

Comparison of protein mixtures in aqueous humor by membrane preconcentration - capillary electrophoresis - mass spectrometry.

The significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1- and 2-dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on-line capillary electrophoresis - mass spectrometry (CE-MS) for this application. Specifically, we used membrane preconcentration-CE-MS (mPC-CE-MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1 microL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS-PROT database using TagIdent from ExPaSy. Among those proteins tentatively identified are beta-2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (beta-2 microglobulin), 13400-14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and approximately 68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the alpha-and beta-chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC-CE-MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids.

Protein analysis by membrane preconcentration-capillary electrophoresis: systematic evaluation of parameters affecting preconcentration and separation.

Fast and efficient analysis of proteins in physiological fluids is of great interest to researchers and clinicians alike. Capillary electrophoresis (CE) has proven to be a potentially valuable tool for the separation of proteins in specimens. However, a generally acknowledged drawback of this technique is the limited sample volumes which can be loaded onto the CE capillary which results in a poor concentration limit of detection. In addition, matrix components in samples may also interfere with separation and detection of analytes. Membrane preconcentration-CE (mPC-CE) has proved to be effective in overcoming these problems. In this report, we describe the systematic evaluation of parameters affecting on-line preconcentration/clean-up and separation of protein mixtures by mPC-CE. Method development was carried out with a standard mixture of proteins (lysozyme, myoglobin, carbonic anhydrase, and human serum albumin). First, using MALDI-TOF-MS, membrane materials with cation-exchange (R-SO3H) or hydrophobic (C2, C8, C18, SDB) characteristics were evaluated for their potential to retain proteins in mPC cartridges. Hydrophobic membranes were found most suitable for this application. Next, all mPC-CE analysis of protein samples were performed in polybrene coated capillaries and parameters affecting sample loading, washing and elution, such as the composition and volume of the elution solvent were investigated. Furthermore, to achieve optimal mPC-CE performance for the separation of protein mixtures parameters affecting postelution focusing and electrophoresis, including the composition of the background electrolyte and a trailing stacking buffer were varied. Optimal conditions for mPC-CE analysis of proteins using a C2 impregnated membrane preconcentration (mPC) cartridge were achieved with a background electrolyte of 5% acetic acid and 2 mM ammonium acetate, 60 nl of 80% acetonitrile in H2O as an elution solvent, and 60 nl of 0.5% ammonium hydroxide as a trailing stacking buffer. The developed method was used successfully to separate proteins in aqueous humor, which contains numerous proteins in a complex matrix of salts.

The analysis of fountain pen inks by capillary electrophoresis with ultraviolet/visible absorbance and laser-induced fluorescence detection.

Information on the identity of inks adds to the circumstantial evidence in legal cases involving fraudulent documents. In combination with optical methods, multiple thin-layer chromatography (TLC) is currently the analytical tool used by forensic chemists to separate, compare and distinguish inks based on their dye composition. In our studies, capillary electrophoresis (CE) was used for the analysis of water-soluble fountain pen inks. Inks are complex mixtures of synthetic organic and inorganic dyes, surfactants, resins and other components. The investigations included the development of an electrophoretic separation method, the optimization of an extraction procedure for inks from paper as well as the evaluation of ultraviolet/visible (UV/VIS) absorbance and laser-induced fluorescence (LIF) detection for the analysis of inks by CE. Good results for the separation of 17 blue and black inks of different manufacturers and countries of origin were obtained with 100 mM borate buffer, pH 8.0, containing 20% methanol. The electropherograms of the inks and their extracts from paper showed patterns that were in most cases distinctly different from each other. Ultraviolet/visible scans can be used to compare spectra of the separated main and trace components of inks. Fluorescence detection at different excitation and emission wavelengths was more sensitive, but added to the complexity of the electropherograms due to the excitation of coextracted fluorescing paper components. The resolution power of CE combined with the information content provided by the detection modes investigated prove CE to be a powerful tool for the identification of water-soluble inks used on paper documents.

Cytotoxicity of spin trapping compounds.

Spin trapping compounds are used frequently to detect free radicals released by cells. Their cytotoxicity has to be considered in order to prevent perturbations of normal cell growth and viability. Eleven spin traps (eight nitrones and three nitroso traps) have been tested for their effects on bovine aortic endothelial cells (toxicity range, 50% survival rate). The lowest cytotoxicity was found for 5,5-dimethylpyrroline-1-oxide and 2,2,4-trimethyl-2H-imidazole-1-oxide whereas nitrosobenzene and 2-methyl-2-nitrosopropane exerted the strongest cytotoxic effects. In addition, three nitronyl nitroxides were tested. Their cytotoxicity was found to be dependent on substitution, and the toxic concentration of a lipophilic derivative was found to be more than two orders lower as compared to a hydrophilic derivative. The results of this study indicate that most spin traps can be used in cell cultures at customary (i.e. millimolar) concentrations; caution is recommended when nitroso spin traps are applied to cells.

Corticotropin-releasing hormone (CRH) receptors in the mesenteric small arteries of rats resemble the (2)-subtype.

The potencies of the corticotropin-releasing hormone (CRH) agonistic peptides oCRH, h/rCRH, frog sauvagine, and carp urotensin I and of the antagonistic peptide alpha-helical CRH9-41 were compared in 3 different in vitro assays: (a) receptor binding to rat brain membranes; (b) release of ACTH/beta-endorphin from rat pituitary cells; and (c) relaxation of rat mesenteric small arteries. From their potency profiles, especially from the high potency of sauvagine relative to CRH in the relaxation assay, it is concluded that the receptors mediating the hypotensive action of systemic CRH in vascular smooth muscle are different from those in the pituitary and brain, and may be identical or very similar to the recently cloned new CRH receptor type 2.

[Sarcoid tumor of the breast]. / Sarkoid-Tumor der Mamma.

We report on a 56 years old women with an enlarging breast mass in the region of an old scar due to a former diagnostic excision. Mammography and sonography were without value while MRT was suspicious for breast cancer. In the frozen section of the excised tissue a carcinoma was diagnosed and therefore a mastectomy performed. This diagnosis was in correct, instead a granulomatous process in the breast and the axillary lymph nodes took place. In respect to a pulmonary sarcoidosis and to laboratory results a true breast sarcoidosis may be supposed. It was also discussed whether it could be a lipogranulomatous pseudosarcoidosis.

The sera of spontaneously hypertensive rats contain agonistic auto-antibodies against the beta 1-adrenoceptor.

OBJECTIVE: To examine auto-antibodies in the sera of spontaneously hypertensive rats (SHR) and investigate the possibility of abnormalities of the immune system in those rats. DESIGN: Blood samples were taken from 18-month-old SHR and age-matched Wistar-Kyoto (WKY) rats. The immunoglobulin fraction was prepared from the rat sera. METHODS: Immunoglobulins were investigated in a sensitive biological test system using spontaneously beating neonatal rat heart myocytes. RESULTS: Immunoglobulins prepared from the sera of the 18-month-old SHR increased the beating frequency of cultured rat heart myocytes. This positive chronotropic effect induced by the auto-antibody-containing immunoglobulin fraction was realized via the beta 1-adrenergic receptor. CONCLUSIONS: The sera of the investigated ageing SHR contain agonistic auto-antibodies directed against the beta 1-adrenoceptor. These anti-beta-adrenoceptor antibodies in the SHR, like those in the sera of cardiomyopathic patients, recognize in most cases the second extracellular loop of the beta 1-adrenoceptor as the site of the antigenic determinant, and act in a similar way to the antibodies observed in the sera of cardiomyopathic patients. The present findings provide the opportunity of using ageing SHR as a model to investigate the development and possible pathogenic role of the agonistic auto-antibodies that recognize the beta 1-adrenoceptor.

4-Hydroxynonenal, a novel indicator of lipid peroxidation for reperfusion injury of the myocardium.

4-Hydroxynonenal (HNE) has been proposed as an important marker of radical-induced lipid peroxidation (LPO) during postischemic reperfusion injury of the myocardium. Therefore, the liberation of HNE into the effluent of isolated perfused rat hearts was investigated. For the first time, the formation of the aldehyde is demonstrated in myocardium. During control perfusion, 1.28 +/- 0.33 pmol HNE.min-1.mg protein-1 were formed by the hearts of 18-mo-old Wistar-Kyoto (WKY) rats and 2.74 +/- 1.12 pmol.min-1.mg protein-1 by those of 18-mo-old spontaneously hypertensive (SHR) rats, respectively. In the WKY group, HNE release increased to 3.35 +/- 1.13 pmol.min-1.mg protein-1 2 min after the onset of reperfusion following 30 min of total and global ischemia compared with the preischemic control period (P < 0.05). In the SHR group, HNE liberation was higher during reperfusion (8.66 +/- 1.33 pmol.min-1.mg protein-1, maximum at 2 min reperfusion) compared with both the respective preischemic control and the respective reperfusion interval of the WKY group (P < 0.05 each). The SHR rats showed signs of congestive cardiac failure of a decompensated hypertrophy in comparison to the normotensive WKY rats. Moreover, the SHR rat hearts exhibited a lower release of adenine nucleotide degradation products (adenine, inosine, hypoxanthine plus uric acid: 48.1 +/- 10.2 nmol.30 min-1.mg protein-1; P < 0.05) and a diminished functional recovery (left ventricular developed pressure, 32 +/- 16 mmHg; P < 0.05) during 30 min of reperfusion compared with the WKY group (77.9 +/- 14.4 nmol.30 min-1.mg protein-1; 90 +/- 21 mmHg).(ABSTRACT TRUNCATED AT 250 WORDS)

[Histology and cytology of laparoscopically operated "simple ovarian cysts"]. / Histologie und Zytologie laparoskopisch operierter "simpler Ovarialzysten".

In a retrospective study the histopathological findings of 127 laparoscopically operated unilocular anechoic smooth-walled ovarian cysts have been correlated with clinical characteristics (age, duration of observation, complaints, hormonal treatment), size by ultrasound, kind and colour of cysts content as well as cytological findings. The age of patients differed from 16-61 years (mean +/- s: 36 +/- 16). The histopathologic findings yielded 15 (11.8%) functional cysts, 30 (23.6%) persistent corpus luteum cysts, 9 (7.1%) endometriomas, 7 (5.5%) cystic teratomas, 9 (7.1%) undifferenciated cysts and 57 (44.9%) cystadenomas. There were no differences between histopathologic diagnosis groups according to age and cysts size by ultrasound. Functional cysts with complaints (n = 6) may explain that the observation time in 60% of all functional cysts was smaller than 6 weeks, whereas persistent corpus luteum cysts, endometriomas, cystic teratoma and cystadenomas had been observed for longer than 6 weeks in more than two thirds. Intraoperative evaluation of cysts content as "chocolate"-like was suspicious of endometriomas, but was also present in cysts of other histopathological findings. By means of cytology, endometrioma (siderophages) was suspected in 44.4% and a cystadenoma in 42.1% of all histopathologically verified cases. In all, the cytologic findings were useful for correct histopathological diagnosis in only 33.9% of all 127 cases. It is concluded that differential diagnosis of simple ovarian cysts is not possible by clinical characteristics, neither by ultrasound nor by cytological evaluation. Ovarian cysts should be observed for at least two hormonal cycles. A hormonal treatment by combination preparations containing high doses of oestrogen is also recommended. In cases of persisting ovarian cysts laparoscopic removal is necessary.