Liliana Andrade

Intravenous leiomyomatosis of the uterus. A report of three cases.

Three cases of intravenous leiomyomatosis (IVL) of the uterus, a rare benign smooth-muscle tumor, are described. A preoperative diagnosis of IVL was not made in any of the patients, all of which presented with a pelvic mass with the presumptive diagnosis of leiomyoma. Surgical exploration confirmed the presence of uterine mass and two of the three cases showed extra-uterine extension into the ovarian or uterine veins. Histological examination demonstrated a fascicular pattern of bland spindle-shaped smooth-muscle cells, which extended to veins inside the myometrium or to extrauterine veins. This was confirmed by immunohistochemical stain for desmin and factor VIII. Despite their histological benignity, these lesions have a tendency to metastasize and are closely related to the conditions called “benign metastasizing leiomyoma” and “intracaval mass and cardiac extension”. The primary treatment of IVL is hysterectomy and excision of any extrauterine tumor, when technically feasible. Anti-estrogenic therapy has been suggested as potentially useful in controlling of unresectable tumor. According to the literature, the follow-up must be long and periodic postoperative ultrasonic or magnetic nuclear resonance imaging studies may be useful in detecting growth of residual intravascular tumor.

The effect upon the human vaginal histology of the long-term use of the injectable contraceptive Depo-Provera®☆

The objective of the study was to evaluate the effect of long-term use of the injectable contraceptive depot medroxyprogesterone acetate (DMPA) on human vaginal histology. Twenty premenopausal women currently using DMPA as a contraceptive method for two and three years were compared with 20 regularly menstruating women, who never used Depo-Provera and/or other kind of hormonal contraceptive in the last 6 months prior to the study. Subjects and controls were matched by age (+/-1 year), body mass index (kg/m2) (+/-1.0), number of pregnancies (+/-1), age at first intercourse (+/-1 year), years of sexual activity (+/-1 year), and number of partners during their life (+/-1). Vaginal biopsies were performed in users at 90+/-7 days after the last injection and in nonusers at day 20-25 of the menstrual cycle. In addition, at the day of the biopsy a blood sample was collected to measure estradiol (in all women) and DMPA in users. The level of serum estradiol was significant lower in Depo-Provera users than in controls (p < 0.001). The thickness of the vaginal epithelium was not smaller among DMPA users than among controls, the mean count of Langerhans cells per mm of epithelium were almost identical in both groups, and no significant differences were found on the vaginal maturation indices. In conclusion, the use of Depo-Provera between two and three years did not affect vaginal thinning of the epithelium, Langerhans cell count or maturation index.

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.

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.

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.

HPV in situ hybridization signal patterns as a marker for cervical intraepithelial neoplasia progression

OBJECTIVE HPV-DNA integration is one factor for malignant transformation and can be identified using in situ hybridization (ISH), where a diffuse signal represents episomal HPV and punctate, integrated. The aim is to verify if a punctate pattern could be a marker of CIN1 that progresses. METHODS 74 CIN1 biopsies were studied. In the follow up, a second biopsy was performed and 65% showed CIN1 or no lesion (group without progression) and 35% CIN2/3 (with progression). ISH was carried out with HR-HPV GenPoint in the first biopsy looking for the positive distribution in epithelium regions (basal, intermediate, superficial) and reaction pattern (diffuse and punctate). The Mann-Whitney and Fisher tests were used to compare the groups (p<or=0.05). RESULTS The mean age of patients without progression was 26 and, with progression 31 (p=0.02). ISH was positive in 22 cases, 8 with CIN2/3 in the second biopsy. The punctate signal was observed in all epithelial layers, and the mean coefficient between the number of cells with punctate and diffuse signals was 3.5 times more common in the progression group (p=0.08). The average percentage of punctate nuclei patterns in the basal region in cases without progression was 0.5% and 11% in those with progression (p=0.05). However, in superficial layer this was not correlated with progression. CONCLUSION Progression was observed in 35% of CIN1 and associated with age; 30% of the cases were positive by ISH, but must be carefully interpreted. Punctate signals were related to progression only in basal cells, identifying CIN1 with potentially aggressive behavior.

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.

Human vaginal histology in long-term users of the injectable contraceptive depot-medroxyprogesterone acetate.

OBJECTIVES Apparently, depot-medroxyprogesterone acetate (DMPA) increases a womans risk of acquiring HIV. The objective of this study was to test whether the vaginal mucosal thickness and Langerhans cell counts were significantly different in long-term DMPA users compared with women users of an intrauterine device (IUD) who had never used DMPA. STUDY DESIGN Cross-sectional study. Twenty-three DMPA users were matched with 23 nonusers controlled for age, body mass index (BMI; kg/m²), and duration of contraceptive use. Four groups of women were evaluated according to the duration of DMPA use: >1, <5; ≥5, <10; ≥10, <15 or ≥15 years. Estradiol (E₂) levels were compared between the two groups. Histologic sections of vaginal mucosal biopsies were evaluated to measure the mean epithelial thickness and S100 immunostained sections were used to count the number of Langerhans cells/mm. RESULTS Mean (±S.D.) E₂ levels were significantly lower in DMPA users (39.4±26.6 pg/mL) compared with nonusers (102.6±60.3 pg/mL) despite similar ages (42.3±7.4 and 42.4±7.4 years, respectively). Mean (±S.D.) vaginal thickness was 232.6±108.1 and 229.7±112.9 in DMPA users and nonusers, respectively. There were no differences in vaginal thickness or Langerhans cell count/mm between users and nonusers even after controlling for DMPA duration of use. CONCLUSIONS Vaginal epithelial thinning or Langerhans cell count was not different between long-term DMPA users and copper-IUD users who had never used DMPA. IMPLICATIONS No differences were found in vaginal epithelial thickness or in Langerhans cell count between long-term users of the injectable contraceptive DMPA and nonusers.

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. Microsc. Res. Tech. 2012.

Prevalence of endometrial cancer and hyperplasia in non-symptomatic overweight and obese women

Background:   Obesity is a public health problem and it is necessary to identify if non‐symptomatic obese women must be submitted to endometrial evaluation.