Epithelial Ovarian Cancer Diagnosis of Second-Harmonic Generation Images: A Semiautomatic Collagen Fibers Quantification Protocol.

A vast number of human pathologic conditions are directly or indirectly related to tissular collagen structure remodeling. The nonlinear optical microscopy second-harmonic generation has become a powerful tool for imaging biological tissues with anisotropic hyperpolarized structures, such as collagen. During the past years, several quantification methods to analyze and evaluate these images have been developed. However, automated or semiautomated solutions are necessary to ensure objectivity and reproducibility of such analysis. This work describes automation and improvement methods for calculating the anisotropy (using fast Fourier transform analysis and the gray-level co-occurrence matrix). These were applied to analyze biopsy samples of human ovarian epithelial cancer at different stages of malignancy (mucinous, serous, mixed, and endometrial subtypes). The semiautomation procedure enabled us to design a diagnostic protocol that recognizes between healthy and pathologic tissues, as well as between different tumor types.

Second harmonic generation microscopy as a powerful diagnostic imaging modality for human ovarian cancer.

In this study we showed that second-harmonic generation (SHG) microscopy combined with precise methods for images evaluation can be used to detect structural changes in the human ovarian stroma. Using a set of scoring methods (alignment of collagen fibers, anisotropy, and correlation), we found significant differences in the distribution and organization of collagen fibers in the stroma component of serous, mucinous, endometrioid and mixed ovarian tumors as compared with normal ovary tissue. This methodology was capable to differentiate between cancerous and healthy tissue, with clear cut distinction between normal, benign, borderline, and malignant tumors of serous type. Our results indicated that the combination of different image-analysis approaches presented here represent a powerful tool to investigate collagen organization and extracellular matrix remodeling in ovarian tumors.

Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy.

We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.

Optical biomarkers of serous and mucinous human ovarian tumor assessed with nonlinear optics microscopies.

BACKGROUND: Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. METHODOLOGY/PRINCIPAL FINDINGS: We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. CONCLUSIONS/SIGNIFICANCE: NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging.

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two-photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy-to-operate platform capable to perform two-photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples.

Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy.

We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG∕THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium∕stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.

Respostas ao adoecimento: mecanismos de defesa utilizados por mulheres com síndrome de Turner e variantes / The defenses employed by women with Turner syndrome: dealing with the disease

OBJETIVO:Conhecer as defesas utilizadas por mulheres com síndrome de Turner (ST) ou formas variantes para lidar com a doença. MÉTODO: Pesquisa qualitativa com desenho exploratório, não experimental. O instrumento consistiu em entrevista psicológica semidirigida, aplicada em 13 mulheres, cuja amostragem deu-se por saturação, as quais fazem acompanhamento semestral no Centro de Atendimento Integral à Saúde da Mulher. Os dados foram interpretados utilizando-se da abordagem psicodinâmica, aliada a um quadro eclético de referenciais teóricos para discussão no espírito da interdisciplinaridade. RESULTADOS: Essas mulheres apresentaram conflitos psicossociais como dificuldades de relacionamento interpessoal; sentimentos de resignação, raiva, impotência, desvalia e quadros de depressão. As defesas utilizadas foram: repressão, negação, anulação, fantasia, adaptação e sublimação. CONCLUSÕES:As mulheres com ST ou formas variantes têm de lidar com as intercorrências orgânicas e psíquicas da enfermidade provocando grande sofrimento que, frequentemente, dificultam uma inserção social mais sadia. Neste caso, os achados deste estudo poderão nortear acompanhamento psicológico ambulatorial concomitantemente ao protocolo clínico de rotina.

OBJECTIVE:To understand the defenses employed by women suffering from Turner syndrome (TS) and different ways of dealing with the disease. METHOD: Qualitative research with exploratory design, non-experimental. The instrument consisted of semi-conducted psychological interview, involving 13 women, undergoing semestral medical follow-up at the Women's Health Care Center, and whose sampling was determined by saturation. Data was interpreted using the psychodynamic approach along with an eclectic framework of theoretical references for discussion in the spirit of interdisciplinary approach. RESULTS:These women displayed psychosocial conflicts such as difficulties in interpersonal relationships; feelings of resignation, anger, impotence, devaluation and depression symptoms. Defenses used were: repression, denial, annulment, fantasizing, adaptation and sublimation. DISCUSSION: Women suffering from TS or variant forms must deal with the disease's organic and psychic implications that cause great suffering and often hinder a healthier social insertion. In this case, the study's findings can guide ambulatory psychological support concomitantly to the routine clinical protocol.

Dental Health, Periodontal and Buccal Hygiene Condition of a Group of Brazilian Women with Breast Cancer

Introduction: The condition of buccal health of patients with cancer may adversely affect the course of treatment, demanding well established conducts of buccal pretreatment through dental evaluations and stabilization of buccal problems. Objective: To evaluate the condition of buccal health prior to chemotherapy, in a group of women with breast cancer assisted in a hospitalof the public system of Campinas, São Paulo. Subjects and methods: In the period 2005-2007, informative lectures were carried out on indications of preventive and curative odontological treatment. Among women interested, 43 having breast cancer were referred to the Service of Dentistry of the institution and evaluated according to the dental condition and need of dental treatment index (DMFT); Community Periodontal Index of Treatment Needs (CPI) and Oral Hygiene Index Simplified. Results and discussion: The mean age of assisted women was 52.5 years, and 70% had tumors in clinical stages II and III. In odontological evaluation, 9.5% of the participants had deep periodontal pockets and 60% showed a regular level of oral hygiene; DMFT was 23.5 teeth and 51% belonged to the women who had their last consultation more than 3 years ago. Due to the scarce number of teeth, there was a reduced volume of odontological procedures to be done. Conclusion: Subjects presented low predominance of dental caries and advanced periodontal disease due to the great number of absent teeth, demanding only low complexity odontological control procedures. Oral hygiene condition was regular.

Immunohistochemical patterns for alpha- and beta-catenin, E- and N-cadherin expression in ovarian epithelial tumors.

OBJECTIVES: This study aims were to analyze and compare the E- and N-cadherin, beta- and alpha-catenin expression in benign and malignant epithelial neoplasms of the ovary, correlating with tumor staging, histological grade, and presence of metastases during evolution. METHODS: Immunohistochemical reactions were performed on paraffin-embedded tissues and evaluated according to the number of positive, stained cellular structures and reaction intensity for each molecule. Information about histological type and grade, tumoral stage, and disease evolution was obtained from the patients' clinical records. RESULTS: Most of the carcinomas showed more intense beta-catenin reaction (P = 0.02). More than 50% of the endometrioid carcinomas showed increased beta-catenin expression, with a large number of positive cells and more intense staining, being the same also observed for most of the serous benign tumors (P < 0.01). E-cadherin membrane expression was frequently observed in carcinomas without metastasis, whereas cases with metastases in evolution were negative or showing E-cadherin expression only in the cytoplasm (P = 0.04). N-cadherin expression differed according to histological type and grade and alpha-catenin was also related to histological type, but these findings were not conclusive. CONCLUSIONS: Increased beta-catenin expression was more frequent in ovarian carcinomas, especially, but not only, in the endometrioid ones. The maintenance of E-cadherin expression in cellular membrane may be an independent marker of good prognosis in ovarian cancer. New studies about N-cadherin and alpha-catenin and their importance during ovarian carcinogenesis will be required.

Estudo citogenético em 80 casos de amenorréia secundária / Cytogenetic study in 80 cases of secondary amenorrhea

OBJETIVO: investigar as alteraçöes citogenéticas, em sangue periférico, em um grupo de mulheres com queixa de amenorréia secundária. AMOSTRA: 80 mulheres atendidas no setor de ginecologia do CAISM, no período de abril de 1990 a março de 1997. DESENHO: idade inferior a 40 anos e queixa principal de amenorréia por período mínimo de 3 ciclos. Os parâmetros estudados compreenderam as dosagens de FSH e LH, realizadas através da técnica de radioimunoensaio, o estudo cromossômico em sangue periférico, realizado em bandas GTG e as quantificaçöes da cromatina X em sedimento urinário. RESULTADOS: a análise citogenética revelou 73 casos com cariótipo normal 46,XX e valores de cromatina X compatíveis com a normalidade, inclusive em 6 casos com estigmas turnerianois. As alteraçöes observadas compreenderam 4 casos de aneuploidias puras, (47,XXX e 45,X), 2 casos de mosaicismo (45,X/46,XX e 45,X/46,X,isoXq) e um caso de deleçäo em braço longo (46,X,delXq(p22q22.1), todos com valores de cromatina X compatíveis com os resultados de cariótipo. As dosagens de LH e FSH permitiram a divisäo da amostra nos subgrupos normo e hipergonadotróficos, com maior incidência deste último e distribuiçäo aproximadamente eqüitativa de alteraçöes citogenéticas nos mesmos. CONCLUSÄO: no grupo estudado, 9 por cento das mulheres apresentaram amenorréia secundária de origem citogenética. Nestes casos, a instalaçäo do quadro clínico ocorreu com idade inferior a 30 anos, em mulheres com sinais clínicos específicos, como baixa ou alta estatura e graus variados de hipodesenvolvimento dos caracteres sexuais secundários. Os valores de cromatina sexual, embora compatíveis com os resultados de cariótipo, mostratam-se ineficazes na detecçäo de mosaicismo e/ou alteraçöes estruturais do cromossomo X. Os autores salientam a necessidade de investigaçäo citogenética de sangue periférico nas mulheres com manifestaçäo de amenorréia secundária com idade inferior a 30 anos e, nos casos de infertilidade por falência ovariana de origem desconhecida e cariótipo 46,XX, a investigaçäo de mosaicismo gonadal.

Seleçäo sexual em dois processos de capacitaçäo espermática: Percoll e Swim-up / Sex selection in human spermatozoa selected by Percoll Gradient centrifugation and Swim-up procedure

OBJETIVO: Investigar a atuaçäo de dois métodos de capacitaçäo espermática como possíveis facilitadores da seleçäo sexual in vitro. DESENHO: foram avaliados os métodos de swim-up de migraçäo em gradiente de Percoll, através da comparaçäo das razöes de sexo obtidas antes e após cada procedimento. A avaliaçäo foi realizada através da visualizaçäo do Corpo F em esfregaços obtidos de suspensöes espermáticas representativas de cada situaçäo, corados pelo diicloreto de quinamicina e observados sob microscopia de fluorescência, empregando-se nas comparaçöes estatísticas o test t de Student e a análise de variância para dados emparelhados. AMOSTRA: doadores saudáveis, com perfil seminal compatível com os níveis de normalidade. RESULTADOS: os dados obtidos indicaram enriquecimento de 15 por cento favorável ao sexo feminino, nas amostras submetidas ao processo de swim-up de 45 minutos. CONCLUSÃO: a capacitaçäo espermática realizada pelo método de Percoll näo introduziu alteraçöes significativas na razäo de sexo gamética. A seleçäo sexual obtida através do swim-up, embora significativa, näo caracterizou uma populaçäo celular altamente purificada.