Laboratory methods in the study of endometrial Claudin-4.

Immunohistochemistry is a suitable method for the detection of proteins from the Claudin family and several antibodies are commercially available for the detection of Claudin congeners. Immunodetection of Caludin-4 in the paraffin-embedded specimens might be a useful tool for studying the role of these proteins in the cyclic transformation of the endometrium and its role in the endometrial receptivity; furthermore, other components of the junctional zone involved in the transformational process of the endometrium can be detected by means of immunohistochemistry/immunofluorescence with several polyclonal or monoclonal antibodies. The aim of this chapter is to comprehensively overview the materials and methods to perform the endometrial biopsy and to detect Claudin-4 in paraffin-embedded samples of endometrium. Additionally, the interpretation of the results is addressed.

Microfluidic culture of single human embryonic stem cell colonies.

We have developed a miniaturized microfluidic culture system that allows experimentation on individual human embryonic stem cell (hESC) colonies in dynamic (flow applied) or static (without flow) conditions. The system consists of three inlet channels that converge into a cell-culture channel and provides the capability to spatially and temporally deliver specific treatments by using patterned laminar fluid flow to different parts of a single hESC colony. We show that microfluidic culture for 96 h with or without flow results in similar maintenance of hESC self-renewal, the capability to differentiate into three germ cell lineages, and to maintain a normal karyotype, as in standard culture dishes. Localized delivery of a fluorescent nucleic acid dye was achieved with laminar flow, producing staining only in nuclei of exposed cells. Likewise, cells in desired regions of colonies could be removed with enzymatic treatment and collected for analysis. Re-coating the enzyme treated area of the channel with extracellular matrix led to re-growth of hESC colonies into this region. Our study demonstrates the culture of hESCs in a microfluidic device that can deliver specific treatments to desired regions of a single colony. This miniaturized culture system allows in situ treatment and analysis with the ability to obtain cell samples from part of a colony without micromanipulation and to perform sensitive molecular analysis while permitting further growth of the hESC colony.

Protein profile of the luteal phase endometrium by tissue microarray assessment.

To investigate the luteal phase endometrial expression of leukemia inhibitor factor (LIF), insulin-like growth factor 1 (IGF-1), progesterone receptor (PR), claudin 4 (CLDN4), vascular-endothelial growth factor receptor 3 (VEGFR-3), bone morphogenetic protein 4 (BMP-4) and citokeratin 7 (CK-7), we obtained luteal phase endometrial samples from 52 women. Samples were dated and integrated using a tissue microarray (TMA). Samples were immunostained for LIF, IGF-1, PR, CLDN4, VEGFR-3, BMP-4 and CK-7. Frequencies of positive expressions at the early, mid and late luteal phases were compared by two proportions test. Concomitant expression of these proteins was assessed with Chi-square or Fischer's test. The frequency of LIF was positively correlated to the frequency of IGF-1 (r = 0.99; p < 0.05) and PR (r = 0.99; p < 0.05), and the correlation between IGF-1 and PR tended to be significant (r = 0.98; p < 0.1). The expression of PR was associated with the absence of CLDN4 (p < 0.001). Thus, expression of LIF, IGF-1 and PR are correlated during the luteal phase, and immunohistochemistry for these proteins might be used to assist in the assessment of endometrial maturation. In addition, the expression of CLDN4 and PR was not concomitant, warranting further investigation on the relationship of their endometrial expression.