Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography.

High-pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 microm, the tubing protects small and fragile samples within the thickness constraints of high-pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.

Characterizing discrete subsets of polycystic ovary syndrome as defined by the Rotterdam criteria: the impact of weight on phenotype and metabolic features.

CONTEXT: The Rotterdam criteria for polycystic ovary syndrome (PCOS) defines discrete subgroups whose phenotypes are not yet clear. OBJECTIVE: The phenotypic characteristics of women in the PCOS subgroups defined by the Rotterdam criteria were compared. DESIGN: The study was observational. SETTING: Subjects were studied in an outpatient setting in Boston and Reykjavik. PATIENTS: Four subgroups of subjects with PCOS defined by 1) irregular menses (IM), hyperandrogenism (HA), and polycystic ovary morphology (PCOM, n = 298); 2) IM/HA (n = 7); 3) HA/PCOM (n = 77); and 4) IM/PCOM (n = 36) and a group of controls (n = 64), aged 18-45 yr, were examined. INTERVENTION: Subjects underwent a physical exam; fasting blood samples for androgens, gonadotropins, and metabolic parameters; and a transvaginal ultrasound. MAIN OUTCOME MEASURES: The phenotype was compared between groups. RESULTS: Ninety-seven percent of women with IM/HA had PCOM. Therefore, the groups with and without PCOM were combined. The Ferriman-Gallwey score and androgen levels were highest in the hyperandrogenic groups (IM/HA and HA/PCOM), whereas ovarian volume was higher in all PCOS subgroups compared with controls, as expected based on the definitions of the PCOS subgroups. Body mass index and insulin levels were highest in the IM/HA subgroup. CONCLUSIONS: Subjects with PCOS defined by IM/HA are the most severely affected women on the basis of androgen levels, ovarian volumes, and insulin levels. Their higher body mass index partially accounts for the increased insulin levels, suggesting that weight gain exacerbates the symptoms of PCOS.

Defining constant versus variable phenotypic features of women with polycystic ovary syndrome using different ethnic groups and populations.

CONTEXT: The phenotype of women with polycystic ovary syndrome (PCOS) is variable, depending on the ethnic background. OBJECTIVE: The phenotypes of women with PCOS in Iceland and Boston were compared. DESIGN: The study was observational with a parallel design. SETTING: Subjects were studied in an outpatient setting. PATIENTS: Women, aged 18-45 yr, with PCOS defined by hyperandrogenism and fewer than nine menses per year, were examined in Iceland (n = 105) and Boston (n = 262). INTERVENTION: PCOS subjects underwent a physical exam, fasting blood samples for androgens, gonadotropins, metabolic parameters, and a transvaginal ultrasound. MAIN OUTCOME MEASURES: The phenotype of women with PCOS was compared between Caucasian women in Iceland and Boston and among Caucasian, African-American, Hispanic, and Asian women in Boston. RESULTS: Androstenedione (4.0 +/- 1.3 vs. 3.5 +/- 1.2 ng/ml; P < 0.01) was higher and testosterone (54.0 +/- 25.7 vs. 66.2 +/- 35.6 ng/dl; P < 0.01), LH (23.1 +/- 15.8 vs. 27.6 +/- 16.2 IU/liter; P < 0.05), and Ferriman Gallwey score were lower (7.1 +/- 6.0 vs. 15.4 +/- 8.5; P < 0.001) in Caucasian Icelandic compared with Boston women with PCOS. There were no differences in fasting blood glucose, insulin, or homeostasis model assessment in body mass index-matched Caucasian subjects from Iceland or Boston or in different ethnic groups in Boston. Polycystic ovary morphology was demonstrated in 93-100% of women with PCOS in all ethnic groups. CONCLUSIONS: The data demonstrate differences in the reproductive features of PCOS without differences in glucose and insulin in body mass index-matched populations. These studies also suggest that measuring androstenedione is important for the documentation of hyperandrogenism in Icelandic women. Finally, polycystic ovary morphology by ultrasound is an almost universal finding in women with PCOS as defined by hyperandrogenism and irregular menses.

Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.

A comprehensive protein resource for the study of bladder cancer: http://biobase.dk/cgi-bin/celis.

In our laboratories we are exploring the possibility of using proteome expression profiles of fresh bladder tumors (transitional cell carcinomas, TCCs; squamous cell carcinomas, SCCs) and random biopsies as fingerprints to subclassify histopathological types and as a starting point to search for protein markers that may form the basis for diagnosis, prognosis, and treatment. Ultimately, the goal of these studies is to identify signaling pathways and components that are affected at various stages of bladder cancer progression and that may provide novel leads in drug discovery. Here we present our ongoing efforts to establish comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) databases of TCCs and SCCs which are being constructed based on the proteomic and immunohistochemical analysis of hundreds of fresh tumors, random biopsies and cystectomies received shortly after operation (http://biobase.dk/cgi-bin/celis).