Detecting microcalcifications in atherosclerotic plaques by a simple trichromic staining method for epoxy embedded carotid endarterectomies.

Atherosclerotic plaques have a high probability of undergoing rapid progression to stenosis, becoming responsible of acute coronary syndrome or stroke. Microcalcifications may act as enhancers of atherosclerotic plaque vulnerability. Considering that calcifications with a diameter smaller than 10 mm in paraffin embedded tissue are rather difficult to detect, our aim was to analyze microcalcifications on semithin sections from epoxy resin embedded samples of carotid endarterectomies using an original trichromic stain (methylene blue--azur B--basic fuchsine--alizarin red). We have compared samples stained either with our method, methylene blue-azur B alone or with Von Kossa staining, and methylene blue-azur B -basic fuchsine alone or with Von Kossa staining. Our method resulted to be simple and fast (ca. 2 min), it gives a sharp general contrast for all structures and allows to easy identify collagen and elastin. In addition, gray-green colour associated to intracellular lipid droplets evidences foam cells, which are particularly abundant in endarterectomies samples. Mast cells and their metachromatic granules are also well recognized. Calcifications over 0,5 mm are clearly recognizable. In conclusion, microcalcifications are clearly distinguished from the extracellular matrix in spite of their reduced dimensions. Methylene blue--azur B--basic fuchsine--alizarin red method is easy to use, reproducible, and is particularly suitable for the identification of microcalcifications in the morphological analysis of atherosclerotic plaques.

Microplicae-like structures of the fallopian tube in postmenopausal women as shown by electron microscopy.

Female reproductive aging is associated with several morphological changes of the genital tract with a subsequent decline in fertility; however, ultrastructural changes occurring after menopause have still not been well illustrated. Our aim was to characterize the three-dimensional microanatomy of the luminal surface of the human fallopian tube in perimenopause and postmenopause. Twenty bioptic samples of fallopian tubes were obtained after surgery under the informed consent of the patients. Samples were processed for transmission electron microscopy (TEM) and field-emission scanning electron microscopy (FE-SEM). As age increases the surface epithelium of the fallopian tube appeared somewhat flattened. Correlated TEM/FE-SEM observations showed gradual shortening of microvilli and deciliation. The most interesting finding was the gradual formation of microplicae-like structures on the surface epithelium, particularly from late perimenopause to postmenopause. Microplicae-like structures, associated with other regressive changes, represent an important adaptation of the epithelium of the fallopian tube; these are likely induced by the physiological process of aging, thus better withstanding hormonal changes associated with the advent of the menopause.

Cumulus oophorus extracellular matrix in the human oocyte: a role for adhesive proteins.

A viscous elastic matrix secreted by the cumulus oophorus cells represent the "extracellular matrix" surrounding the human mature oocyte obtained from assisted reproductive technology (ART). The cumulus matrix is involved in several reproductive processes, including the pick-up of the oocyte-cumulus complex by the oviduct, the final maturation of the ovum and sperm-egg interaction. As showed by some Authors, the cumulus matrix is rich in hyaluronan, as well as in other proteins including inter-alpha-trypsin inhibitor, a dermatan sulfate proteoglycan, and a pentraxin-3. Proteins and hyaluronan are linked together to form a meshwork comprised of granules and filaments. We found in human cumulus oocyte complexes the presence of specialized cells still capable of producing fibronectin and tenascin-c in the post-ovulatory period. Moreover, fibronectin and tenascin-c are present within the hyaluronan matrix at fertilization during the tubal sojourn of the ovum and the embryo. Since pentraxin3 is important in matrix stabilization and gamete interactions, a possible role for fibronectin and tenascin-c may be postulated in the final maturation of the ovum, in the tubal pick-up and in the complex dialogue with the tubal epithelium.

The human zona pellucida and scanning electron microscopy. Reality or artifacts?

The surface micro-morphology of the zona pellucida (ZP) was investigated in 158 inseminated but unfertilized mature human oocytes derived from assisted reproduction trials (ART) by means of traditional scanning electron microscopy (SEM) techniques (gold coating and conductive staining methods) and saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide method (Sap-RR-Os-TC). The main aspect of the ZP by traditional SEM (122 oocytes) consisted in a porous, net-like structure (97 oocytes), whereas a nearly smooth or compact structure of ZP was detected in 25 oocytes (79.5% vs 20.5%). Using Sap RR-Os-TC method on 36 oocytes, 31 oocytes showed ZP with alternating tight and large meshed networks, whereas 5 oocytes displayed only tight meshed network (86.1% vs 13.9%). Due to our well standardized procedures, to the stabilizing action of the conductive staining on the zona material and similar results obtained with the use of Sap RR-Os-TC method, we confidentially regard the ZP changes, occurring in oocytes of various groups, as genuine features, likely related to their actual maturation status, rather than as artifacts. In addition, we emphasize the concept that a modern view of the ZP surface implies the best evidence of crossing filaments' network. We think that the ZP "spongy" or "compact" appearance is only the result of microfilaments network collapse, not the true three-dimensional (3-D) representation of ZP structure.