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Background Diagnosis of thyroid dysfunction relies on thyroid stimulating hormone (TSH), free thyroxine (FT4), and free tri-iodothyronine (FT3) tests against valid reference intervals (RIs). We changed the immunoassay platform from Abbott Architect to Siemens Atellica and aimed to establish Atellica RIs based on laboratory information system (LIS) patient data. Methods Atellica thyroid hormone immunoassays were verified against those of Architect. Real-life patient results were retrieved from LIS. A single result per patient dataset was used to establish the RIs by the indirect method. Results Atellica and Architect assays correlated well but Atellica showed a positive bias between 13% and 53%, the largest for FT4. Variations of the Atellica assays were ≤4%. The 95% Atellica RIs were 0.4–3.8 mU/L for TSH, 0.9–1.6 ng/dL for FT4, and 227–416 pg/dL for FT3. Considering the accumulating clinical experience with Atellica, the RIs for clinical use were adjusted as 0.5–4.0 mU/L, 0.9–1.8 ng/dL, and 169–409 pg/dL, respectively. Conclusions We verified thyroid hormone RIs for Atellica by the indirect method for the first time. Our model proved reliable for selecting results of presumably healthy individuals from LIS data. Critical review of the RIs with local endocrinologists is essential.

A recurrent mutation in FOXL2 (c.402C>G; p.C134W) is present in over 95% of adult-type granulosa cell tumours (AGCTs). In contrast, various loss-of-function mutations in FOXL2 lead to the development of blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). BPES is characterised by an eyelid malformation often accompanied with primary ovarian insufficiency. Two recent studies suggest that FOXL2 C402G is a gain- or change-of-function mutation with altered DNA-binding specificity. Another study proposes that FOXL2 C402G is selectively targeted for degradation, inducing somatic haploinsufficiency, suggesting its role as a tumour suppressor. The latter study relies on data indicative of an FOXL2 allelic imbalance in AGCTs. Here we present RNA-seq data as genetic evidence that no real allelic imbalance is observed at the transcriptomic level in AGCTs. Additionally, there is no loss of protein expression in tumours harbouring the mutated allele. These data and other features of this mutation compared to other oncogenes and tumour suppressor genes argue strongly against FOXL2 being a tumour suppressor in this context. Given the likelihood that FOXL2 C402G is oncogenic, targeting the variant protein or its downstream consequences is the most viable path forward to identifying an effective treatment for this cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objectives There are many mobile health applications (apps) now available and some that use in some way laboratory medicine data. Among them, patient-oriented are of the lowest content quality. The aim of this study was to compare the opinions of non-laboratory medicine professionals (NLMP) with those of laboratory medicine specialists (LMS) and define the benchmarks for quality assessment of laboratory medicine apps. Methods Twenty-five volunteers from six European countries evaluated 16 selected patient-oriented apps. Participants were 20–60 years old, 44% were females, with different educational degrees, and no professional involvement in laboratory medicine. Each participant completed a questionnaire based on the Mobile Application Rating Scale (MARS) and the System Usability Scale, as previously used for rating the app quality by LMS. The responses from the two groups were compared using the Mann-Whitney U test and Spearman correlation. Results The median total score of NLMP app evaluation was 2.73 out of 5 (IQR 0.95) compared to 3.78 (IQR 1.05) by the LMS. All scores were statistically significantly lower in the NLMP group (p Conclusions NLMP’ evaluation confirmed the low utility of currently available laboratory medicine apps. A reliable app should contain trustworthy and understandable information. The appearance of an app should be fit for purpose and easy to use.

Objectives Teaching involves multiple performance situations, potentially causing psychosocial stress. Since the theater-based improvisation method is associated with diminished social stress, we investigated whether improvisation lessened student teachers’ stress responses using the Trier Social Stress Test (TSST; preparatory phase, public speech, and math task). Moreover, we studied the influence of interpersonal confidence (IC) – the belief regarding one’s capability related to effective social interactions – on stress responses. Methods The intervention group (n = 19) received a 7-week (17.5 h) improvisation training, preceded and followed by the TSST. We evaluated experienced stress using a self-report scale, while physiological stress was assessed before (silent 30-s waiting period) and during the TSST tasks using cardiovascular measures (heart rate, heart rate variability [HRV]), electrodermal activation, facial electromyography (f-EMG), and EEG asymmetry. Hypothalamus-pituitary-adrenal (HPA-axis) reactivity was assessed through repeated salivary cortisol sampling. Results Compared to the control group (n = 16), the intervention group exhibited less f-EMG activity before a public speech and higher HRV before the math task. The low IC intervention subgroup reported significantly less stress during the math task. The controls showed a decreased heart rate before the math task, and controls with a low IC exhibited higher HRV during the speech. Self-reported stress and cortisol levels were positively correlated during the post-TSST preparatory phase. Conclusions These findings suggest that improvisation training might diminish stress levels, specifically before a performance. In addition, interpersonal confidence appears to reduce stress responses. The decreased stress responses in the control group suggest adaptation through repetition.

Adult-type granulosa cell tumors (AGCTs) are sex-cord derived neoplasms with a propensity for late relapse. Hormonal modulators have been used empirically in the treatment of recurrent AGCT, albeit with limited success. To provide a more rigorous foundation for hormonal therapy in AGCT, we used a multimodal approach to characterize the expressions of key hormone biomarkers in 175 tumor specimens and 51 serum samples using RNA sequencing, immunohistochemistry, RNA in situ hybridization, quantitative PCR, and circulating biomarker analysis, and correlated these results with clinical data. We show that FSH receptor and estrogen receptor beta (ERβ) are highly expressed in the majority of AGCTs, whereas the expressions of estrogen receptor alpha (ERα) and G-protein coupled estrogen receptor 1 are less prominent. ERβ protein expression is further increased in recurrent tumors. Aromatase expression levels show high variability between tumors. None of the markers examined served as prognostic biomarkers for progression-free or overall survival. In functional experiments, we assessed the effects of FSH, estradiol (E2), and the aromatase inhibitor letrozole on AGCT cell viability using 2 in vitro models: KGN cells and primary cultures of AGCT cells. FSH increased cell viability in a subset of primary AGCT cells, whereas E2 had no effect on cell viability at physiological concentrations. Letrozole suppressed E2 production in AGCTs; however, it did not impact cell viability. We did not find preclinical evidence to support the clinical use of aromatase inhibitors in AGCT treatment, and thus randomized, prospective clinical studies are needed to clarify the role of hormonal treatments in AGCTs.

Adult granulosa cell tumors (AGCTs) of the ovary are molecularly characterized by the pathognomonic FOXL2 402C>G (C134W) mutation. To improve diagnostics and follow-up, we developed a specific digital droplet PCR (ddPCR) assay to detect the FOXL2 mutation in the circulating tumor DNA (ctDNA) of AGCT patients. Optimization of the ddPCR assay was performed using a TaqMan primer/probe with the RainDance RainDrop digital PCR system. The ddPCR assay was performed on circulating cell-free DNA extracted from 120 serial plasma samples collected prospectively from 35 AGCT patients. The ddPCR assay included a preamplification step that is sensitive and specific for detecting the FOXL2-mutated ctDNA at levels as low as 0.05%. FOXL2 ctDNA mutations were detected in the plasma of 12 of 33 AGCT patients (36%), with both primary (6 of 17, 35%) and recurrent (6 of 31, 19%) tumors. The median tumor size was significantly larger in ctDNA mutation–positive compared with mutation-negative samples (13.5 cm versus 7.5 cm; P = 0.003). The ctDNA FOXL2 mutation was detected in four patients without clinical disease, of which one relapsed during follow-up. As proof of concept, we established that specific molecular diagnosis of AGCT and detection of AGCT recurrence can be achieved noninvasively using ctDNA FOXL2 mutation testing. Further studies are needed to determine the clinical value of ctDNA mutation testing.

Objective. Resistance to standard chemotherapy poses a major clinical problem in the treatment of ovarian cancer patients. Adult-type granulosa cell tumor (AGCT) is a unique ovarian cancer subtype for which efficient treatment options are lacking in advanced disease. To this end, systematic drug response and transcriptomics profiling were performed to uncover new therapy options for AGCTs. Methods. The responses of three primary and four recurrent AGCTs to 230 anticancer compounds were screened in vitro using a systematic drug sensitivity and resistance testing (DSRT) platform, coupled with mRNA sequencing. The responses of the AGCTs were compared with those of human granulosa luteal cells and bone marrow mononuclear cells. Results. Patient-derived AGCT cells showed selective sensitivity to the Src family tyrosine kinase inhibitor dasatinib. A combination of either dasatinib or an mTOR-inhibitor everolimus with paclitaxel resulted in synergistic inhibition of AGCT cell viability. The key kinase targets of dasatinib and members of the mTOR pathway were constantly expressed at mRNA and protein levels, indicating multikinase signal addictions in the AGCT cells. Transcriptomic characterization of the tumors revealed no known oncogenic mutations, suggesting that the drug sensitivity of AGCTs was rather conveyed by selective target expression. Conclusions. We used a systematic functional approach to reveal novel treatment options for a unique gynecological cancer. The selective synergy found between taxanes and dasatinib or mTOR inhibitors warrants further clinical investigations of these combinations in relapsed or aggressive AGCTs and demonstrate that high throughput drug screening and molecular profiling can provide an effective approach to uncover new therapy options. (C) 2016 Elsevier Inc. All rights reserved.

In the future, boreal forests will be growing in a warmer climate with more fluctuating soil moisture conditions. However, the knowledge about the effects of simultaneous warming and drought on boreal trees, and especially on their belowground compartments, is still scarce. We studied the responses of four silver birch genotypes to experimental warming at normal and low watering levels after two growing seasons. Variables analysed include mycorrhizal short root growth, fungal biomass (ergosterol) in roots and extramatrical mycelium, and root morphology (root tissue density, root length density, specific root length, specific root surface area). In addition, root and shoot mass fractions were determined. Gt26 had more root mass and also more 2–5 mm diameter roots, but less short roots and ergosterol in its roots than the other genotypes. There were no treatment effects on extramatrical mycelium and mycorrhizal infection levels, but root ergosterol concentrations increased in gt12 due to warming. Drought increased root mass at the expense of shoot growth in gt12, but warming did not cause allometric growth changes in any of the genotypes. Our results show some genotype-dependent responses to warming and drought but also significant within population differences in belowground allocation patterns among the silver birch genotypes.

Differences in the response to adverse environmental conditions among genotypes within local populations are important in determining the ability of local populations to cope with such conditions, especially for long-lived organisms like trees. Nevertheless, such differences and their relevance for acclimation have not been addressed to date, representing a gap in our understanding of tree growth and acclimation. Focussing on temperature and soil moisture we address this issue, evaluating 13 relevant physiological (i.e. gas exchange, leaf pigments) and leaf morphological traits (e.g. specific leaf area) and three traits related to growth partitioning for two growing seasons in 10 genotypes of the ecologically and economically important tree species silver birch (Betula pendula Roth). Complex trait syndromes – a consistent association of plant traits characterising differences among genotypes – including physiological, leaf morphological and growth partitioning traits were identified. The relative investment in leaf mass, shoot-to-root ratio and net assimilation rate were most important for good growth. With regard to acclimation to adverse environmental conditions genotypes mostly acclimated in a similar way, but changes in allocation patterns may depend on genotype, even within a local population. Trees with a high net assimilation rate and a low leaf mass fraction and shoot-to-root ratio produced most biomass after two years of growth. Information on these traits could, in addition to growth, be useful in identifying material for forest regeneration that is robust with regards to expected climate change.

Ovarian adult-type granulosa cell tumors (AGCTs) require prolonged follow-up, but evidence regarding the optimal follow-up marker is lacking. The objective of our study was to validate the clinical usefulness of serum anti-Mullerian hormone (AMH) and the current marker inhibin B as single and combined markers of AGCTs. We conducted a longitudinal, partially prospective cohort study of 123 premenopausal and postmenopausal AGCT patients with a median follow-up time of 10.5 years (range 0.3-50.0 years). Serum AMH and inhibin B levels were measured from 560 pretreatment and follow-up serum samples by using immunoenzymometric assays. We found that serum AMH and inhibin B levels were significantly elevated in patients with primary or recurrent AGCTs. The levels of both markers positively correlated to tumor size (p

Within a local population genotypes differ in the timing of bud burst, but genotypes with early bud burst unfold their leaves slower, resulting in an equal period of carbon gain. The ability of local populations to cope with disturbances like adverse weather events or a changing climate depends on the genotypic richness of such populations, emphasising the importance of differences between genotypes in traits related to growth and survival at this scale. Due to their longevity, these differences are of special importance in trees, yet for trees, differences between genotypes within local populations remain unexplored. The phenological cycle is important in this respect, since a correct timing of phenological events is critical for growth and survival of trees, especially in environments with strong seasonality and changes in the timing of phenological events has consequences for, among others, net ecosystem productivity and the climate system as a whole. In this light accounting for differences in the timing of phenological events within species is currently identified as a research challenge. This study contributes to the knowledge of differences between genotypes on the small spatial scale of a local population. We examined the timing of phenological events of 15 micropropagated silver birch (Betula pendula Roth) genotypes representing a natural population. Measurements covered bud burst (7 years) and leaf unfolding in spring and chlorophyll degradation in autumn (2 years for both). These data were used to estimate the period of carbon gain. Differences between genotypes in the temperature sum required for bud burst were present, with genotypes showing ‘early’ (i.e. a low temperature sum requirement for bud burst) and ‘late’ bud burst across the 7-year study period. Differences were small in most years (i.e. 3 days), but differences of 16 days were recorded within the 7-year study period as well. Genotypes with ‘early’ bud burst were less sensitive to variations in environmental conditions in spring compared to genotypes with ‘late’ bud burst. Differences in bud burst were not carried over to the estimated period of carbon gain. Due to faster leaf expansion in genotypes with ‘late’ bud burst and the lack of differences between genotypes in autumn senescence the estimated period of carbon gain was similar among genotypes.

Granulosa cell tumors (GCTs) carry a risk of recurrence also at an early stage, but reliable prognostic factors are lacking. We assessed clinicopathological prognostic factors and the prognostic roles of the human epidermal growth factor receptors (HER 2-4) and the transcription factor GATA4 in GCTs. We conducted a long-term follow-up study of 80 GCT patients with a mean follow-up time of 16.8 years. A tumor-tissue microarray was immunohistochemically stained for HER2-4 and GATA4. Expression of HER2-4 mRNA was studied by means of real time polymerase chain reaction and HER2 gene amplification was analyzed by means of silver in situ hybridization. The results were correlated to clinical data on recurrences and survival. We found that GCTs have an indolent prognosis, with 5-year disease-specific survival (DSS) being 97.5%. Tumor recurrence was detected in 24% of the patients at a median of 7.0 years (range 2.6-18 years) after diagnosis. Tumor stage was not prognostic of disease-free survival (DFS). Of the molecular prognostic factors, high-level expression of HER2, and GATA4, and high nuclear atypia were prognostic of shorter DFS. In multivariate analyses, high-level coexpression of HER2 and GATA4 independently predicted DFS (hazard ratio [HR] 8.75, 95% CI 2.20-39.48, P = 0.002). High-level expression of GATA4 also predicted shorter DSS (HR 3.96, 95% CI 1.45-12.57, P = 0.006). In multivariate analyses, however, tumor stage (II-III) and nuclear atypia were independent prognostic factors of DSS. In conclusion HER2 and GATA4 are new molecular prognostic markers of GCT recurrence, which could be utilized to optimize the management and follow-up of patients with early-stage GCTs.

Hintergrund: Die histopathologische Diagnostik adulter Granulosazelltumoren (aGCT) beruht auf der Begutachtung morphologischer und immunhistochemischer Kriterien. Aufgrund bislang nicht verfugbarer tumorspezifischer Marker kann die Begutachtung entsprechender Tumoren auch fur den erfahrenen Pathologen in manchen Fallen eine Herausforderung darstellen. Im Jahre 2009 wurde eine fur aGCT spezifische Mutation im FOXL2-Gen beschrieben. Ziel dieser Arbeit war es die Bedeutung der FOXL2-Mutationstestung in einem grosen Kollektiv ursprunglich als aGCT diagnostizierter Tumoren zu untersuchen. Material und Methoden: Etablierung eines Studienkollektives bestehend aus 336 adulten Granulosazelltumoren durch Rekrutierung in drei gynako-onkologischen Zentren (Amsterdam, Helsinki, Tubingen). Samtliche Originaldiagnosen wurden gemas morphologischer und immunhistochemischer Kriterien gestellt. FOXL2-Mutationsanalyse mittels TaqMan Assay, abschliesend spezialisierte Zweitbegutachtung samtlicher FOXL2-negativer Falle. Ergebnisse: Bestatigung der Originaldiagnose „aGCT“ durch Nachweis der FOXL2-Mutation in 76% aller Falle (256/336). In den verbleibenden 24% wurden 63/80 (79%) aller Originaldiagnosen revidiert (Andere Keimstrang-Stromatumore: n = 32; Epitheliale Tumore: n = 29; Andere: n = 2). Bei 72% aller krankheitsbedingten Todesfalle handelt es sich in den ersten 5 Jahren nach Diagnose um Falle mit revidierter Originaldiagnose. Fur das Gesamtuberleben konnte zwischen der Gruppe mit bestatigter Diagnose „aGCT“ (trotz Rezidiven in 28% aller Falle) und einer altersangepassten Kontrollgruppe kein statistisch signifikanter Unterschied gezeigt werden. Diskussion: Samtliche bis zum heutigen Tage verfugbaren Daten und Therapieempfehlungen zur Diagnose „adulter Granulosazelltumor“ beruhen unter Berucksichtigung aktueller Forschungsergebnisse hochstwahrscheinlich auf Studienkollektiven, in denen bis zu 20% falschlicherweise als aGCT diagostizierte Falle enthalten waren. Vor diesem Hintergrund mussen bisherige Erkenntnisse kritisch hinterfragt werden, insbesondere sollte das Potential rein chirurgischer Therapiekonzepte und die auch in der Rezidivsituation gute Prognose der Erkrankung berucksichtigt werden.

The histopathologic features of adult granulosa cell tumors (AGCTs) are relatively nonspecific, resulting in misdiagnosis of other cancers as AGCT, a problem that has not been well characterized. FOXL2 mutation testing was used to stratify 336 AGCTs from three European centers into three categories: 1) FOXL2 mutant molecularly defined AGCT (MD-AGCT) (n = 256 of 336), 2) FOXL2 wild-type AGCT (n = 17 of 336), 3) misdiagnosed other tumor types (n = 63 of 336). All statistical tests were two-sided. The overall and disease-specific survival of the misdiagnosed cases was lower than in the MD-AGCTs (P < .001). The misdiagnosed cases accounted for 71.9% of disease-specific deaths within five years. In the population-based cohort, overall survival of MD-AGCT patients was not different from age-matched, population-based controls. Even though 35.2% of all the MD-AGCT patients in our study experienced a relapse, AGCT is usually an indolent disease. The historical, premolecular data underpinning our clinical understanding of AGCT was likely skewed by inclusion of misdiagnosed cases, and future management strategies should reflect the potential for surgical cure and long survival even after relapse.

Ovarian granulosa cell tumors (GCT) are highly vascularized and express vascular endothelial growth factor A (VEGF) and its functional receptor VEGF receptor 2 (VEGFR-2). Angiogenesis inhibitors have been used in the treatment of ovarian carcinomas, whereas their roles in GCT remain unknown.The aim was to assess serum levels of VEGF and endostatin, an endogenous angiogenesis inhibitor, in GCT patients and to study the effect of bevacizumab (BVZ), a VEGF-binding monoclonal antibody, on human GCT cells in vitro.Using ELISA, we measured soluble VEGF and endostatin in the sera of 54 GCT patients and in conditioned media from cultures of 14 primary GCT and an established GCT cell line (KGN). The expression of activated VEGFR-2 was analyzed in GCT tissues using immunohistochemistry. GCT cells were treated with BVZ and analyzed for cell number and apoptosis.Serum VEGF was elevated in GCT patients, and the levels significantly decreased after tumor removal (P0.05), whereas serum endostatin levels changed conversely. Human GCT expressed activated VEGFR-2 protein, and the level of expression was associated with tumor VEGF and vascularization. In addition, the cultured GCT cells produced significant amounts of VEGF but not endostatin. Treatment of KGN cells with BVZ significantly reduced the number of viable cells by 41% and induced a 3.3-fold increase in apoptosis. Furthermore, BVZ induced a mean 2.6-fold increase in apoptosis in six primary GCT cell cultures studied.These data suggest an autocrine role for VEGF in GCT and encourage clinical studies on anti-VEGF treatments in this disease.

FOXL2 is a transcription factor that is essential for ovarian function and maintenance, the germline mutations of which are responsible for the Blepharophimosis Ptosis Epicanthus-inversus Syndrome (BPES), often associated with premature ovarian failure. Recent evidence has linked FOXL2 downregulation or somatic mutation (p.Cys134Trp) to cancer, although underlying molecular mechanisms remain unclear. Using a functional genomic approach, we find that FOXL2 modulates cell-cycle regulators in a way which tends to induce G1 arrest. Indeed, FOXL2 upregulation promotes cell accumulation in G1 phase and protects cells from oxidative damage, notably by promoting oxidized DNA repair and by increasing the amounts of anti-oxidant agent glutathione. In agreement with clinical observations, we find that FOXL2-mutated versions leading to BPES along with ovarian dysfunction mostly fail to transactivate cell-cycle and DNA repair targets, whereas mutations leading to isolated craniofacial defects (and normal ovarian function) activate them correctly. Interestingly, these assays revealed a mild promoter-specific hypomorphy of the tumor-associated mutation (p.Cys134Trp). Finally, the SIRT1 deacetylase suppresses FOXL2 activity on targets linked to cell-cycle and DNA repair in a dose-dependent manner. Accordingly, we find that SIRT1 inhibition by nicotinamide limits proliferation, notably by increasing endogenous FOXL2 amount/activity. The body of evidence presented here supports the idea that FOXL2 plays a key role in granulosa cell homeostasis, the failure of which is central to ovarian ageing and tumorigenesis. As granulosa cell tumors respond poorly to conventional chemotherapy, our findings on the deacetylase inhibitor nicotinamide provide an interesting option for targeted therapy.

Crowberry (Empetrum nigrum L.) is a relatively under-utilized wild berry that occurs widely throughout the northern hemisphere such as in Canada, Eurasia, and northern Europe. In this work, the anthocyanins of crowberries were analyzed from four geographically distinct crowberry populations in Finland using HPLC-DAD and HPLC-ESI/MS/MS. A total number of 15 anthocyanins were detected; 15 (11 structure elucidated) in all samples in order to profile-specific anthocyanin compositions throughout Finland. The major anthocyanin found in the samples collected from central and eastern Finland was delphinidin 3-galactoside accounting for more than 24% of the total anthocyanin content, while the cyanidin 3-galactoside was the major anthocyanin in the northernmost and in the western samples. Significant variation in the concentrations of different anthocyanins between and within crowberry populations were found suggesting that the synthesis of anthocyanins is modified by site-specific environmental conditions. The suitability of the crowberries as a potential source of health-promoting ingredients for incorporation into pharmaceutical and food industrial products is highlighted in this work due to the diverse anthocyanin profile.

Granulosa cell tumors of the ovary represent ∼5% of malignant ovarian cancers. It has recently been reported that 95-97% of adult granulosa cell tumors carry a unique somatic mutation in the FOXL2 gene. We undertook this study to verify the presence of the FOXL2 Cys134Trp mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of FOXL2 in these tumors. A total of 56 tumors with the histological diagnosis of adult granulosa cell tumor from two centers, Melbourne and Helsinki, were examined for the presence of the mutation using direct sequence analysis. Two granulosa cell tumor-derived cell lines, COV434 and KGN, three juvenile granulosa cell tumors and control tissues were also examined. The expression of the FOXL2 gene was determined using quantitative RT-PCR and/or immunohistochemistry. We found that 52 of the 56 adult granulosa cell tumors harbor the mutation, of which three were hemi/homozygous. Of the four cases with wild-type FOXL2 sequence, reappraisal suggests that three may have been misclassified at primary diagnosis. The KGN cells were heterozygous for the mutation, whereas the COV434 cells had a wild-type FOXL2 genotype. The expression levels of FOXL2 were similar across the adult granulosa cell tumors and the normal ovary controls; one mutation-negative granulosa cell tumor had high FOXL2 mRNA levels, whereas the COV434 cells and two of the three juvenile granulosa cell tumors lacked the expression of FOXL2. Our data provide confirmation of the frequent presence of the FOXL2 C134W mutation in adult granulosa cell tumors and demonstrate that the mutation is not associated with altered FOXL2 expression. The mutation analysis may be a useful tool to differentiate particularly between cell-rich diffuse granulosa cell tumors and mitotically active sex cord-stromal tumors. This unique FOXL2 mutation appears to be characteristic of adult granulosa cell tumors.

Comparative targeted compositional analysis is currently an important element in the safety assessment of genetically modified plants. Profiling methods have been suggested as nontargeted tools to improve the detection of possible unintended effects. In this study, the capability of 2-dimensional electrophoresis to detect significant differences among seven conventional maize (Zea mays) cultivars grown in six different locations in Germany during two consecutive seasons was evaluated. Besides maize genotype, both geographic location and season had a significant effect on protein profiles. Differences as high as 55- and 53-fold in the quantity of specific proteins were recorded, the median observed difference being around 6- and 5-fold between the genotypes and growing locations, respectively. Understanding the variation in the quantity of individual proteins should help to put the variation of endogenous proteins and the novel proteins in the genetically modified plants in perspective. This together with the targeted analyses the profiling methods, including proteomics, could also help to get a deeper insight into the unintended alterations that might have occurred during the genetic modification process.

The anti-Müllerian hormone (AMH) belongs to the TGF-β family and plays a key role during fetal sexual development. It has been shown that AMH inhibits proliferation and induces apoptosis of AMH type II receptor (AMHRII) -positive tumor cells, especially progenitor cancer cells. Moreover, various reports have described the expression of AMHRII in human gynecologic cancers including ovarian tumors. Based on qRT-PCR test results, which were confirmed by a specific In-Situ Hybridization (ISH) assay, AMHRII mRNA could be detected only in normal ovary, testis and adrenal gland tissue, and at lower levels in pancreas and stomach. However, when a tissue microarray of solid tumor samples was tested with ISH assay, AMHRII mRNA was surprisingly detected in several primary cancer tissues including melanoma, lung, head and neck, colorectal and breast cancers. For example, a large ISH study involving 58 colorectal cancer samples demonstrated AMHRII expression in 55% of cases. AMHRII protein expression was confirmed by ImmunoHistoChemistry (IHC) in gynecological cancer samples including 179 out of 210 (85%) Granulosa Cell Tumor samples and 55 out of 80 (69 %) Epithelial Ovarian Cancers. In patients with gynecological cancers pre-screened by IHC for inclusion in a Phase Ia/Ib clinical trial of GM102, an anti-AMHRII monoclonal antibody, AMHRII positive membrane expression was detected in 116 out of 166 (70%) archived tumor tissues. Using the same IHC protocol, over 1000 frozen samples covering 16 different cancer types were tested, which demonstrated AMHRII expression in over 50% of hepatocarcinoma, colorectal, lung and renal cancer samples. No AMHRII expression was observed in 75 neuroendocrine lung tumor samples nor in 18 non-Hodgkin lymphoma samples tested. On a small panel of frozen ovarian (11) and colorectal (9) cancer samples using an immunofluorescence assay with an AlexaFluor488-conjugated GM102 antibody, AMHRII expression was confirmed on the matching fresh tissue samples, previously detected by IHC in their fixed counterpart samples. Preliminary FACS analysis of fresh ovarian and colorectal tumor samples have demonstrated comparable expression levels with mean values of 50,000 and 40,000 AMHRII receptors per cell, respectively. In conclusion, the results presented above, using a variety of detection methods and systems, have consistently shown that AMHRII is expressed in many different histological types of solid tumors. These results suggest that this embryonic receptor could be a suitable target for selective antitumor agents such as GM102. Citation Format: Jean-Marc M. Barret, Didier Meseure, Guillaume Bataillon, Noora Andersson, Aurélie Auguste, Olivier Dubreuil, Anniina Färkkilä, Emeline Perrial, Celine Bossard, Emily Loison, Anne Jarry, André Nicolas, Fanny Lemée, Solenne Gaillard, Mehdi Lahmar, Mikko Anttonen, Gabriel Champenois, Charles Dumontet, Isabelle Ray-Coquard, Alexandra Leary, Isabelle Tabah-Fisch, Anne Vincent-Salomon, Jean-François F. Prost. Anti-Müllerian hormone type II receptor (AMHRII) found expressed in human non-gynecological solid tumors, suggesting potential broader applications for anti-AMHRII-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 774.

International audience; WNT4 plays an important role in female sexual development and ovarian function. WNT4- deficiency leads disturbed development of the internal genitalia in mouse and human, and to a dramatic reduction of mouse oocytes. However, the expression and role of WNT4 in human ovaries is yet unknown. The expression of WNT4 mRNA and protein was studied in human adult and fetal ovaries (gestational ages 12–41 weeks), and the role of Wnt4 in oocyte apoptosis was investigated in Wnt4-deficient mice. WNT4 mRNA and protein were present in human ovaries throughout fetal development and in different follicular stages in adult ovaries. Compared with wild-type mice, Wnt4-deficient mice had a markedly enhanced rate of oocyte apoptosis, with the highest values at gestational ages of 14.5 and 16.5 days post-coitum. The current results support a view that WNT4 may have a role in oocyte selection and follicle formation and maturation in human ovaries.

P>The aim of this study was to evaluate the use of four nontargeted analytical methodologies in the detection of unintended effects that could be derived during genetic manipulation of crops. Three profiling technologies were used to compare the transcriptome, proteome and metabolome of two transgenic maize lines with the respective control line. By comparing the profiles of the two transgenic lines grown in the same location over three growing seasons, we could determine the extent of environmental variation, while the comparison with the control maize line allowed the investigation of effects caused by a difference in genotype. The effect of growing conditions as an additional environmental effect was also evaluated by comparing the Bt-maize line with the control line from plants grown in three different locations in one growing season. The environment was shown to play an important effect in the protein, gene expression and metabolite levels of the maize samples tested where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed. A distinct separation between the three growing seasons was also found for all the samples grown in one location. Together, these environmental factors caused more variation in the different transcript/protein/metabolite profiles than the different genotypes.

Phenolic compounds form a large group of plant secondary metabolites with many functions related to the acclimation and adaptation of plants to a changing environment and to the interaction with other organisms. Interestingly, numerous studies have shown the positive influence of phenolic compounds on human health, and a higher intake is beneficial. Thus, phenolic compounds can be considered as an important quality factor in soft fruits. In this study the potential of different cultivation practices to increase the content of phenolic compounds in red raspberry (Rubus idaeus), strawberry (Fragaria × ananassa) and black currant (Ribes nigrum) was evaluated. The study shows that several possible ways exist to enhance the content of phenolic compounds in crop plants including the choice of cultivar, fertilization, mulching, light and temperature. However, as different methods have different shortcomings, they should be thoroughly evaluated before their application in practice.

Modification of plants by introducing the stilbene synthase (STS) gene has often resulted in novel production of the phytoalexin resveratrol, and provided enhanced resistance against several pathogenic fungi. In the present study, a grapevine STS expressing strawberry (Fragaria × ananassa) was investigated for the effect of the transgene on plant metabolism by techniques including quantitative real-time PCR, UPLC-qTOF-MS profiling and NMR analysis. The introduced STS gene caused down-regulation of the strawberry endogenous chalcone synthase (CHS) gene expression. The perturbation of the gene expression was reflected at the metabolite content, as the CHS enzyme downstream products, mainly flavonols, were found at reduced levels concomitantly with the accumulation of the precursor molecules like phenolic acid derivatives. In addition, several previously unidentified metabolites were accumulating in the leaves of the transgenic strawberry plants. A detailed metabolite analysis was pertinent for the detection of unintended consequences of the gene transfer, and eventually provided deeper insight in the phenolic compound metabolism of strawberry.

Considerable amount of recent epidemiological data suggest that a high intake of fruits and vegetables offers a number of health benefits against degenerative diseases and can promote longevity. Black currant fruits are particularly rich sources of biologically active compounds, for example high levels of anthocyanins, proanthocyanidins, quercetin, myricetin, phenolic acids, and isorhamnetin are found in black currant. In addition, black currant possesses a high content of vitamin C, contributing together with bioactive phenolics to the high antioxidant activity of berries. Multiple health benefits by black currant phenolics have been suggested by a number of recent studies including the inhibition of development of certain cancers, cardiovascular and inflammation related diseases. Blackcurrant was recently demonstrated to provide effective neuroprotection against oxidative stress induced neuronal damages in human cell cultures. Among phenolics, anthocyanins are considered the most potent neuroprotective compounds found in soft fruits. Black currant also contains a wide range of flavonols including myricetin, quercetin and isorhamnetin, these flavonols have been demonstrated to possess neuroprotective activity. Black currant is in Europe an important berry for the food industry mainly because of its color and organoleptic properties, which makes it a suitable material for diverse food applications. Improving fruit quality by classical breeding methods is of big challenge, and recent advances in the development of molecular markers enables breeders to select complex traits with high accuracy and faster than conventional methods. Improving the levels of health-promoting compounds in black currants by genetic transformation is in an early stage but offers a great potential for black currant improvement in coming years as has been demonstrated in other plants.

Introduction Strawberry (Fragaria × ananassa) is rich in polyphenols, particularly anthocyanins, flavonols, condensed tannins and ellagic tannins. In addition to the fruits, the leaves of strawberry also contain a wide range of phenolic compound classes, but have not been investigated to the same extent as the fruit. Objective To characterise a metabolite group present in the leaves of strawberry, that was not amenable for identification based on earlier information available in the literature. Methodology Methanolic extracts of strawberry leaves were analysed by UPLC-qTOF-MS/MS and iterative quantum mechanical NMR spectral analysis. Results The structures of phenylethanol derivatives of phenylpropanoid glucosides Eutigoside A ( F4) and its two isomeric forms 2-(4-hydroxyphenyl)ethyl-[6-O-(Z)-coumaroyl]-β-d-glucopyranoside ( F6) and 4-(2-hydroxyethyl)phenyl-[6-O-(e)-coumaroyl]-β-d-glucopyranoside ( F1) were resolved by NMR and UPLC-qTOF-MS/MS. In addition, two other derivatives of phenylpropanoid glucosides similar to Eutigoside A but possessing different phenolic acid moieties, namely Grayanoside A ( F5) and 2-(4-hydroxyphenyl)ethyl-[6-O-(e)-caffeoyl]-β-d-glucopyranoside ( F14), were similarly identified. Also, accurate characteristic coupling constants for the subunits are reported and their usefulness in structural analysis is highlighted. Conclusion Chemical analysis of the leaves of strawberry (Fragaria × ananassa cv. Jonsok) resulted in the identification of a compound class, phenylethanol derivatives of phenylpropanoid glycosides, not previously found in strawberry. Copyright © 2009 John Wiley & Sons, Ltd.

Excessive cell proliferation and decreased apoptosis have been implicated in the pathogenesis of ovarian granulosa cell tumors (GCTs). We hypothesized that transcription factor GATA-4 controls expression of the antiapoptotic factor Bcl-2 and the cell cycle regulator cyclin D2 in normal and neoplastic granulosa cells. To test this hypothesis, a tissue microarray based on 80 GCTs was subjected to immunohistochemistry for GATA-4, Bcl-2, and cyclin D2, and the data were correlated to clinical and histopathological parameters. In addition, quantitative RT-PCR for GATA-4, Bcl-2, and cyclin D2 was performed on 21 human GCTs. A mouse GCT model was used to complement these studies. The role of GATA-4 in the regulation of Bcl2 and ccdn2 (coding for cyclin D2) was studied by transactivation assays, and by disrupting GATA-4 function with dominant negative approaches in mouse and human GCT cell lines. We found that GATA-4 expression correlated with Bcl-2 and cyclin D2 expression in human and murine GCTs. Moreover, GATA-4 enhanced Bcl-2 and cyclin D2 promoter activity in murine GCT cells. Whereas GATA-4 overexpression up-regulated and dominant negative GATA-4 suppressed Bcl-2 expression in human GCT cells, the effects on cyclin D2 were negligible. Our results reveal a previously unknown relationship between GATA-4 and Bcl-2 in mammalian granulosa cells and GCTs, and suggest that GATA-4 influences granulosa cell fate by transactivating Bcl-2.

The effects of exposing strawberry plants to a short-day treatment (10 h day-1) from August 10 to September 17, and reducing the crown number per plant in the following spring were examined on fruit yield and quality in Haygrow® (UK) tunnel-grown strawberries. Reducing the number of crowns per plant increased °Brix level and reduced fruit yield. The yield reduction was lower than the reduction in crown number should account for, mainly because the number of fruits per crown was higher on plants with reduced crown number than on multicrown (MC) plants. Short-day treatment increased yield by 10% in MC plants compared with plants grown under ambient light, but the length of the photoperiod had no effect on quality compounds irrespective of crown number. However, for fruit size there was a significant interaction between day-length and crown number, expressed as increased fruit size of plants under ambient light conditions and no effect on short-day plants owing to reduced crown number. There were no...

Black currants (Ribes nigrum L.) contain a diverse range of phenolics and possess a high antioxidant activity, which makes them an interesting target for the functional food industry. In this study, phenolic profiles of organically and conventionally grown black currant fruits, collected from commercial farms within a climatically similar area, were compared. Compounds were identified using UV/vis and mass spectroscopy techniques and quantified with high-performance liquid chromatography equipped with UV/vis detection. Several different conjugates of hydroxycinnamic acids, flavonols, and anthocyanins were quantified. Statistically significant differences between farms were found for almost all compounds. Differences between the highest and the lowest measured values of major phenolic compounds of different phenolic classes ranged from 24 to 77%. Principal component analysis quite effectively separated farms from each other but did not cluster them according to cultivation technique. Thus, it was concluded that the biochemical quality of organically grown black currant fruits does not differ from those grown conventionally.

Part of heterodimeric inhibin, inhibin-α is crucial for mammalian ovarian function. Regulation of inhibin-α expression in granulosa cells is both endocrine, primarily by follicle-stimulating hormone (FSH), and paracrine, primarily by members of the transforming growth factor β (TGF-β) superfamily. Smad proteins transmit TGF-β signals to the nucleus, but the cooperating transcription factors involved in inhibin-α promoter activation remain unknown. Transcription factor GATA-4 regulates inhibin-α in gonadal cells, and the FSH cascade activates GATA-4. We hypothesized that the TGF-β signalling cascade and GATA-4 also cooperate to regulate inhibin-α expression. In KK-1 granulosa tumour cells, which resemble normal granulosa cells and express inhibin-α, we found that TGF-β upregulated GATA-4 expression. Transient transfection experiments in KK-1 cells demonstrated that dominant negative GATA-4 variants or mutations of GATA-binding sites in the inhibin-α promoter attenuated TGF-β-induced gene activation. In GATA-4-deficient COS-7 cells, TGF-β enhanced the expression of the inhibin-α promoter only in the presence of exogenous GATA-4. Smad3, but not Smad2, cooperated with GATA-4 in the transcriptional activation of the inhibin-α promoter, and immunoprecipitation experiments in KK-1 cells revealed a physical Smad3:GATA-4 interaction. Our data suggest that GATA-4, interacting with Smad3, is a cofactor for TGF-β signalling to activate inhibin-α in granulosa cells.

The influence of agricultural practices (fertilization, mulch color, early forcing, and planting date), environment (light and growing area), cultivar, and fruit order on the selected phenolic content and antioxidant activity in strawberry (Fragaria x ananassa Duch.) fruits was studied. Three different levels of fertilization were given to plants in the fertilization experiment. The lowest fertilization level increased the contents of flavonols and ellagic acid from 19 to 57%. Between cultivars, up to 4-fold differences were found in the flavonol content, and it also varied according to growing environment. Planting date in glasshouse production was important for the phenolic content, and a statistically significant interaction was found between planting date and fruit order. Fruit order caused at highest 1.5-2.0-fold differences in the contents of phenolics. Interestingly, compared with other phenolics, anthocyanins were affected differently by many factors. Thus, the findings show that minor cultivation changes can increase the content of phenolics, especially in under-glass production where conditions can be easily manipulated.

Granulosa cell tumors (GCTs) are ovarian malignancies that produce estrogens, inhibins, and anti-Müllerian hormone (AMH). The molecular pathogenesis of GCTs is likely to involve defects in the genes regulating normal granulosa cell proliferation during folliculogenesis.The objective of this study was to test the role of factors regulating the normal granulosa cell function, i.e. AMH, inhibin-alpha, SF-1 (steroidogenic factor-1), and GATA transcription factors in the pathobiology and clinical behavior of GCTs.We selected randomly a cohort of 80 GCT patients treated at our university hospital during 1971-2003, analyzed protein expression in the tumor samples embedded on a tissue microarray by immunohistochemistry, and correlated the data to clinical and histopathological parameters.We found no significant differences in the immunoreactivity levels of inhibin-alpha, GATA-6, FOG-2 (friend of GATA-2), or SF-1 in GCTs compared with normal granulosa cells. AMH expression was, however, low (i.e. reduced) in 69% of GCTs and correlated inversely with tumor size (P = 0.0025). In contrast, GATA-4 expression was high (i.e. resembled normal granulosa cells) in 44% of GCTs and correlated positively with clinical stage and recurrence (P = 0.0232 and P = 0.0038, respectively). Fifty of the 80 patients had a follow-up for at least 10 yr, and 13 of them had recurrence(s). In multivariate analysis of recurrence, the high GATA-4 expression remained the only independent factor (risk ratio, 9.2; 95% confidence interval, 2.0-43.3; P = 0.0048).The more aggressive GCTs retain a high GATA-4 expression, whereas the larger tumors lose the proliferation-suppressing AMH expression. The high GATA-4 expression in GCTs may serve as a marker of poor prognosis.

Red raspberry ( Rubus idaeus L.) is an economically important berry crop that contains numerous phenolic compounds with potential health benefits. It is known that the content of phenolics is affected by processing factors, but limited information is available on the influence of cultural factors or genotype. To clarify this issue, phenolic compounds were analysed from a diverse range of raspberry cultivars grown under northern European conditions, in Finland. The content of phenolic compounds varied widely and significantly between cultivars. The quercetin content ranged from 0.32 (yellow cultivar) to 1.55 mg/100 g fresh weight (fw) (cv. Balder). The ellagic acid content varied from 38 (cv. Gatineau and cv. Nova) to 118 mg/100 g fw (cv. Ville). The total anthocyanin content varied from close to 0 (yellow cultivars) to 51 mg/100 g fw (cv. Gatineau). The content of total phenolics varied from 192 (cv. Gatineau) to 359 mg/100 g fw (cv. Ville). In addition, environment had a significant effect on the content of quercetin. Thus, breeding material should be evaluated for their potential health benefits over several regions in northern raspberry breeding.

Small nuclear RING finger protein (SNURF/RNF4) is a steroid receptor coregulator that is down-regulated in testicular germ cell cancer. In this work, we examined SNURF expression during murine fetal gonad development and postnatal ovarian folliculogenesis by in situ hybridization and immunohistochemical staining. SNURF mRNA was detectable in gonads of both sexes from embryonic 10.5 days post conception onward. SNURF protein localized to gonocytes and somatic Leydig and Sertoli cells of fetal testis and in oogonia and supporting cells of fetal ovary. In murine postnatal ovary, SNURF mRNA and protein were expressed throughout folliculogenesis, peaking in the oocytes of preantral follicles. Lower amounts of SNURF mRNA and protein were also present in granulosa cells of secondary, antral, and preovulatory follicles and in luteal glands. Exposure of immature female mice and rats to gonadotropin from pregnant mare serum and human chorionic gonadotropin did not change dramatically SNURF mRNA levels in ovary. SNURF mRNA expression was increased in ovaries of immature mice treated with diethylstilbestrol, an effect that was blocked by the pure antiestrogen ICI 182,780. SNURF protein was constitutively expressed in oocytes of hypophysectomized rats, and its content was augmented by estradiol in granulosa cells. In granulosa cell culture, SNURF mRNA accumulation was transiently increased by treatment with the LH agonists phorbol myristate and forskolin at 4 h after treatment and at 48 h in differentiated cells expressing markers of the preovulatory phenotype. These results suggest a role for SNURF in fetal germ cell development as well as in oocyte and granulosa cell maturation in an estrogen- and gonadotropin-regulated fashion.

Flavonoids are a large group of over 4000 secondary plant products, comprising anthocyanins, flavonols, flavones, flavanones and catechins. Flavonoids are known to act as protective compounds possessing antioxidant and free radical scavenging activity in foods. An HPLC-based method was used to analyze the content of flavonols in strawberry, raspberry and blackcurrant cultivars grown under northern conditions. Blackcurrants contained about ten times more flavonols than strawberries and raspberries. Quercetin, kaempferol and myricetin were all found in blackcurrants. In strawberries, quercetin and kaempferol, and in raspberries only quercetin was found. High variability in flavonol contents among strawberry, raspberry and blackcurrant cultivars was observed. High variation was also found in flavonol and total phenolic contents in various blackcurrant juices. Analyses of flavonol contents from strawberries and blackcurrant cultivars grown organically or conventionally revealed no significant differences. Genotype seems to be a more important factor than cultivation technique in the accumulation of phenolic compounds in berries. The high variability among berry cultivars offers possibilities to enhance the value of berry production by plant breeding and growing cultivars rich in flavonols.

Plants have developed multiple protection strategies during evolution in response to invading pathogens and environmental stresses. Flavonoids are a large and diverse group of over 4000 compounds, many of which are known to play an important role against UV-light stress and microbial attacks in plants. Many of these plant defence compounds are also health-promoting phytochemicals in the human diet. Strawberry (Fragaria x ananassa) contains a variety of such phenolic compounds. We found that the most disease resistant (to gray mold) strawberry cultivar contained considerably more flavonoids than the most susceptible one, but the role of these compounds in disease resistance is still unclear. Defence mechanisms in plants can be activated by biotic or abiotic elicitors, which trigger a defence pathway leading to the expression of several defence genes and the accumulation of antifungal compounds, which eventually leads to the inhibition of the fungal growth. We tested the ability of benzothiadiazole (BTH) to induce resistance to powdery mildew (Sphaerotheca macularis) in strawberry. Powdery mildew has become a serious disease in strawberry grown in greenhouses in many parts of the world. As chemical control is difficult because of the rapid development of fungicide-insensitive strains, alternative strategies are needed. We showed that foliar applications of BTH (0,6-1,2 g/l) on young strawberry leaves provide effective mildew control in greenhouses. The flavonol glycoside content of BTH-treated leaves was also analyzed by HPLC and ESI-MS method, but no clear differences were detected between BTH-treated and control plants. However, the levels of two flavonols, quercetin and kaempferol, in berries were increased in response to foliar treatments under field conditions. The results suggest that BTH and related bioactivators may become a realistic alternative for chemical control against powdery mildew in strawberries grown in greenhouses and plastie tunnels.

Transcription factor GATA-4 has been suggested to have a role in mammalian gonadogenesis, e.g., through activation of the Mullerian-inhibiting substance (MIS) gene expression. Although the expression of GATA-4 during gonadogenesis has been elucidated in detail, very little is known about FOG-2, an essential cofactor for GATA-4, in ovarian development. We explored in detail the expression of FOG-2 and GATA-4 in the fetal and postnatal mouse ovary and in the fetal testis using Northern blotting, RNA in situ hybridization, and immunohistochemistry. GATA-4 and FOG-2 are evident in the bipotential urogenital ridge, and their expression persists in the fetal mouse ovary; this result is different from earlier reports of GATA-4 downregulation in the fetal ovary. In contrast to ovary, FOG-2 expression is lost in the fetal Sertoli cells along with the formation of the testicular cords, leading to the hypothesis that FOG-2 has a specific role in the fetal ovaries counteracting the transactivation of the MIS gene by GATA-4. In vitro transfection assays verified that FOG-2 is able to repress the effect of GATA-4 on MIS transactivation in granulosa cells. In postnatal ovary, granulosa cells of growing follicles express FOG-2, partially overlapping with the expression of MIS. These data suggest an important role for FOG-2 and the GATA transcription factors in the developing ovary.

OBJECTIVE: The transcription factors GATA-1 and GATA-4 have been implicated in the regulation of testicular development and function. Their cofactors FOG-1 and FOG-2 are expressed in the gonads, but their cell-specific and developmental expression in the testis remains unresolved. Therefore, we analyzed GATA-1, GATA-4, FOG-1 and FOG-2 expression in detail, from undifferentiated male urogenital ridge to adult testis. METHODS: Immunohistochemistry and in situ hybridization were applied on mouse testicular samples. RESULTS: GATA-4 and FOG-2, but not GATA-1 or FOG-1, were expressed as early as in the male urogenital ridge. FOG-2 expression was localized in the Sertoli cells at embryonal day 12.5 (E12.5), but it diminished with advancing fetal testicular development. In E17.5 testis, FOG-2 was present only in the testicular capsule and a subset of fetal Leydig cells. FOG-1 was expressed from E15.5 Sertoli cells onwards, whereas GATA-1 was not detected during the fetal period at all. In the postnatal testis, FOG-2 was abundantly expressed immediately after birth, but in adult testis its expression was predominantly restricted to stage VII-XII seminiferous tubules. Stage specificity was also found for FOG-1, which, similarly to GATA-1, was abundantly expressed in stage VII-XII tubules during adulthood. CONCLUSIONS: Our results indicate that FOG-2, in addition to GATA-4, has a role in early gonadal development and sexual differentiation, and FOG-1 at later fetal stages, while GATA-1 executes its action postnatally. The findings suggest that, in contrast to the hematopoietic system and the heart, GATA-1 and GATA-4 do not use FOG-1 and FOG-2 respectively as their only cofactors during the early stages of testicular development.

Objective: To study the gonadal steroid responses to FSH and hCG in individuals with the inherited Finnish-type inactivating Ala189Val mutation of the FSH receptor gene. Design: Prospective clinical and descriptive study. Setting: University hospital. Patient(s): Two women and one man homozygous for the Ala189Val mutation of the FSH receptor gene, and ovarian biopsies from four affected and four healthy women, and four normal fetuses. Intervention(s): Individuals were treated with increasing doses of recombinant FSH (300 IU/day start, 900 IU/day final) and/or a single dose of hCG (5000 IU). Ovarian biopsies were used in immunohistochemical analyses for detection of aromatase cytochrome P450 and transcription factor GATA-4. In situ 3′-end labeling analyses were used for detection of apoptosis. Main Outcome Measure(s): Measurements of serum concentrations of follicle-stimulating hormone, leuteinizing hormone, inhibin A and B, estradiol, testosterone (T), androstenedione, and prolactin, immunostaining for ovarian aromatase, GATA-4, and apoptosis. Result(s): Administration of FSH had no effect on production of the steroids. Similarly, human chorionic gonadotropin (hCG) treatment, alone or after FSH administration, failed to raise serum steroid concentrations. Ovarian apoptosis was absent, and the expression of transcription factor GATA-4 and aromatase was negligible in the ovarian biopsies from Ala189Val homozygous individuals. Conclusion(s): The Ala189Val mutation of the FSH receptor gene results in a complete block of FSH action in vivo. Furthermore, the failure of hCG to increase both ovarian estradiol and testosterone secretion emphasizes the possible contribution of FSH in regulating ovarian androgen synthesis, and supports the concept that both gonadotropins are necessary for appropriate ovarian steroidogenesis in humans.

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ObjectiveThe objective of this study was to evaluate clinical prognostic factors and survival of patients with ovarian granulosa cell tumors (GCTs) in a long-term follow-up study.MethodsA total of 240 adult-type GCTs diagnosed in Helsinki University Central Hospital from 1956 to 2012 were histologically reevaluated. Data were analyzed for several clinical factors in relation to major developments in imaging, surgery, and chemotherapy: the old era (1956–1983) and the new era (1984–2012). Prognostic factors for survival were evaluated in the univariate and multivariate analyses.ResultsThe original diagnosis was confirmed in 187 (77.9%) patients. The International Federation of Gynecology and Obstetrics stage I disease was present in 89.2%; stage II, in 7.0%; stage III, in 3.8%; and stage IV, in 0% of cases. The mean age at diagnosis (52.9 years) and the mean tumor size (10.8 cm) did not change significantly over time. The most common presenting symptom was abnormal bleeding, but 14% were asymptomatic. The mean follow-up period was 15.7 years. Recurrence rate was similar in both eras. The GCT-specific 5-, 10-, and 20-year survival rates were 95.6%, 88.1%, and 79.8% in the old era as well as 97.2%, 94.8%, and 94.8% in the new era, respectively. In the univariate analyses, old era, patient age older than 60 years, tumor size greater than 10 cm, advanced stage, residual tumor, and use of hormonal adjuvant treatment were associated with GCT-related deaths. Prior use of oral contraceptives and history of infertility improved survival rates. In the multivariate analysis, stage was the only independent prognostic factor for GCT-specific survival.ConclusionsAn accurate histological diagnosis of GCT is essential. Stage IV disease is an extreme rarity. However, tumor stage overcomes other possible clinical prognostic factors for GCT-specific survival. Fertility-sparing surgery, the use of oral contraceptives, or hormonal replacement therapy seems not to be risk factors for survival.

International audience; Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-β mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis.

The majority of oocytes present in fetal ovaries are depleted before birth, and only about 400 will ovulate during the normal fertile life span. Studies on animals have shown that apoptosis is the mechanism behind oocyte depletion and follicular atresia. In the present study, we investigated the extent and localization of apoptosis in human fetal (aged 13–40 weeks) and adult ovaries. Furthermore, the expression of apoptosis-regulating proteins, bcl-2 and bax, and the relationship of transcription factor GATA-4 were studied. Apoptosis was found in ovarian follicles throughout fetal and adult life. During fetal development, apoptosis was localized mainly to primary oocytes and was highest between weeks 14–28, decreasing thereafter toward term. Expression of bcl-2 was observed only in the youngest fetal ovaries (weeks 13–14), and bax was present in the ovaries throughout the entire fetal period. In adult ovaries, apoptosis was detected in granulosa cells of secondary and antral follicles, and Bcl-2 and bax were expressed from primary follicles onwards. During fetal ovarian development, GATA-4 messenger RNA and protein were localized to the granulosa cells, with expression being highest in the youngest ovaries and decreasing somewhat toward term. The expression pattern of GATA-4 suggests that it may be involved in the mechanisms protecting granulosa cells from apoptosis from fetal to adult life. The results indicate that depletion of ovarian follicles in the human fetus occurs through intrinsic mechanisms of apoptosis in oocytes, and later in adult life the survival of growing follicles may be primarily determined by granulosa cell apoptosis.

Using comparative genomic hybridization (CGH), we have previously demonstrated frequent loss of 8p, especially its distal part, in ovarian carcinoma. To compare the deletion map of distal 8p in serous and mucinous ovarian carcinomas, we performed allelic analysis with 18 polymorphic microsatellite markers at 8p21-p23. In serous carcinoma, loss of heterozygosity (LOH) was detected in 67% of the samples, and the majority of the carcinomas showed loss of all or most of the informative markers. In contrast, only 21% of mucinous carcinomas showed allelic loss, with only one or two loci showing LOH in each sample. In serous carcinomas, LOH was associated with higher grade tumors. Three distinct minimal common regions of loss could be defined in serous carcinomas (at 8p21.1, 8p22-p23.1, and 8p23.1). Expression of a transcription factor gene, GATA4, located at one of these regions (8p23.1) was studied in serous and mucinous ovarian carcinomas by Northern blotting and immunohistochemical staining of tumor microarray. Expression was found to be lost in most serous carcinomas but retained in the majority of mucinous carcinomas. Our results suggest distinct pathogenetic pathways in serous and mucinous ovarian carcinomas and the presence of more than one tumor suppressor gene at 8p involved in the tumorigenesis of serous carcinoma.

Previous studies have implicated transcription factors GATA-4 and GATA-6 in the regulation of murine ovarian development and function. In rodents, GATA-4 is expressed in granulosa cells of primary and early antral follicles, whereas GATA-6 is expressed in granulosa cells of late antral follicles and luteal glands. Both transcription factors can be detected in lesser amounts in theca cells and interstitial cells. We have now examined the expression of GATA-4 and GATA-6 in human ovaries, human granulosa-luteal (GL) cells and sex cord-derived tumors. We show by in situ hybridization and immunohistochemistry that GATA-4 and GATA-6 messenger RNA (mRNA) and GATA-4 protein are present in granulosa and theca cells in both preantral and antral follicles. Both human ovarian tissue samples and freshly isolated GL cells derived from preovulatory follicles of gonadotropin-treated women express GATA-4, GATA-6, and FOG-2 transcripts, and GATA-6 mRNA expression in GL cell cultures is stimulated by human CG and 8-bromo-cAMP. The vast majority of granulosa and theca cell tumors examined expressed GATA-4 and GATA-6. We also found that mRNA for FOG-2, a recently discovered regulator of GATA-4, is coexpressed with GATA-4 in human ovary samples, normal granulosa cells, and in sex cord-derived tumors. Our results demonstrate that GATA-4, GATA-6, and FOG-2 are expressed in human ovary and in granulosa and theca cell tumors. Our findings support a role for GATA-binding proteins in human ovarian folliculogenesis. Moreover, these data suggest that GATA factors may contribute to the phenotypes of sex cord-derived ovarian tumors.

Yolk sac tumors (YSTs) are malignant tumors that occur in the gonads of children and young adults, and at extragonadal sites in young children. The histological features of YSTs are variable and can be superimposed on other germ cell tumor histologies. Malignant endodermal cells within YSTs express alpha-fetoprotein, which can be detected in tumor tissue or serum. However, additional markers of endoderm differentiation would be beneficial for the classification of these tumors. Transcription factor GATA-4 regulates the differentiation and function of murine yolk sac endoderm, and its expression correlates with proliferation and cell survival in certain tissues. To see whether GATA-4 plays a role in human YSTs, we surveyed its expression in human germ cell tumors and cell lines. Northern analysis demonstrated expression of GATA-4 mRNA in four human germ cell tumor lines exhibiting yolk sac endoderm differentiation. GATA-4 protein was detected in eight of nine pediatric YSTs by immunohistochemistry. Three of five immature teratomas exhibited GATA-4 in neural blastematous cells and in cylindrical epithelium, whereas all 16 mature teratomas were devoid of GATA-4. We conclude that GATA-4 is a clinically useful marker of human YSTs and speculate that it may play a role in the maintenance of the malignant phenotype.

International audience; Ovarian granulosa cell tumors (OGCT) are the most frequent kind of sex cord-stromal tumors, and represent ∼2-5% of all ovarian malignancies. OGCTs exist as two entities, juvenile and adult types, with specific clinical and pathological characteristics. The molecular pathogenesis of these tumors has just begun to be unraveled. Indeed, recent studies have indicated that mutation and/or misregulation of the key ovarian transcription factor FOXL2 has a role in OGCT formation, although the mechanisms remain unclear. To better understand the molecular characteristics of OGCT, we studied the transcriptomic profiles of ten human adult-type OGCT samples, as well as ethnically matched granulosa cell (GC) controls. We find that the OGCT samples analyzed herein exhibit several hallmarks of cancer, including increased expression of genes linked to cell proliferation, but decreased expression of those conferring sensitivity to cell death. Moreover, genes differentially expressed in OGCTs are significantly enriched for known FOXL2 target genes, consistently with the prevalence of FOXL2 somatic mutation in these tumors. Expression of these targets is altered in a way expected to promote malignant transformation, for instance, through induction of genes associated with faster cell cycling and downregulation of genes associated with cell death. Over time, such defects may be responsible at least partly for the malignant transformation of healthy GCs into OGCT. These insights into the molecular pathogenesis of OGCTs may open the way to new efforts in the development of more targeted therapeutic strategies for OGCT patients.Oncogene advance online publication, 16 July 2012; doi:10.1038/onc.2012.298.

Ovarian granulosa cell tumors (GCTs) are sex cord stromal tumors that constitute 3-5% of all ovarian cancers. GCTs usually present with an indolent course but there is a high risk of recurrence, which associates with increased mortality, and targeted treatments would be desirable. Anti-Mullerian hormone (AMH), a key factor regulating sexual differentiation of the reproductive organs, has been implicated as a growth inhibitor in ovarian cancer. GCTs and normal granulosa cells produce AMH, but its expression in large GCTs is usually downregulated. Further, as the lack of specific AMH-signaling pathway components leads to GCT development in mice, we hypothesized that AMH inhibits growth of GCTs. Utilizing a large panel of human GCT tissue samples, we found that AMH type I receptors (ALK2, ALK3 and ALK6) and type II receptor (AMHRII), as well as their downstream effectors Smad1/5, are expressed and active in GCTs. AMHRII expression was detected in the vast majority (96%) of GCTs and correlated with AMH mRNA and protein expression. AMH mRNA level was low in large GCTs, confirming previous findings on low-AMH protein expression in large human as well as mouse GCTs. To study the functional role of AMH in this peculiar ovarian cancer, we utilized a human GCT cell line (KGN) and 10 primary GCT cell cultures. We found that the AMH-Smad1/5-signaling pathway was active in these cells, and that exogenous AMH further activated Smad1/5 in KGN cells. Furthermore, AMH treatment reduced the number of KGN cells and primary GCT cells, with increasing amounts of AMH leading to augmented activation of caspase-3 and subsequent apoptosis. All in all, these data support the premise that AMH is a growth inhibitor of GCTs.

ObjectiveOvarian granulosa cell tumors (GCTs) are hormonally active sex cord stromal tumors accounting for 3–5% of all ovarian cancers. These tumors are generally diagnosed at an early stage but there is a high risk of recurrence, associated with high mortality. Treatment of recurrent GCTs is difficult, and biologically targeted treatment modalities are lacking. GCTs are highly vascularized, and angiogenic factors most probably play a role in their pathology. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis, but in GCTs, the role of VEGF and its receptors VEGFR-1 (FLT1) and VEGFR-2 (KDR) remains largely unknown. Our objective is to study the expression of VEGF and its receptors in human GCTs.MethodsWe analyzed GCTs from 106 patients for the expressions of VEGF and its receptors utilizing tumor tissue microarray, tumor mRNA, and patient serum samples.ResultsWe found that VEGF and its main biologically active receptor VEGFR-2 were highly expressed in primary and recurrent GCTs, when compared with normal granulosa-lutein cells. The expression of VEGF correlated positively to tumor microvessel density and to VEGFR-2 expression at the protein (PPConclusionsThe results suggest an important role of VEGF and VEGFR-2 in GCT pathology and support the possibility of applying novel VEGF- or VEGFR-2-targeted treatments to patients with GCT.