Usefulness of ploidy, AgNOR and immunocytochemistry for differentiating benign and malignant cells in serous effusions.

OBJECTIVE: The objective of this study was to establish the value of different markers in differentiating reactive mesothelial cells from metastatic adenocarcinomatous cells in serous effusions (SE). METHODS: Forty-five SE were processed for morphological examination (Papanicolaou stain), assessment of ploidy, AgNOR counting and immunocytochemical assay of carcinoembryonic antigen (CEA), epithelial membrane antigens (EMA), Ber-EP4 and Leu-M1. Ploidy was established in an image-analyser in smears stained by the Feulgen stain method. AgNOR dots were counted in the smears stained with the silver nitrate assay for non-histone proteins present in the nucleolar organizer region. CEA, EMA, Ber-EP4 and Leu-M1 were evaluated by immunocytochemistry using the streptavidin-biotin complex method. RESULTS: All the smears with positive cytology were aneuploid. Three false negatives by morphological studies were aneuploid, with AgNOR values in two of them corresponding to the neoplastic group. CEA and Leu-M1 showed a low specificity; EMA and Ber-EP4 showed moderate sensitivity. CONCLUSIONS: The assessment of ploidy and the study of AgNOR were better methods than immunocytochemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid.

Asthenozoospermia: analysis of a large population.

Spermatozoa travel a long distance to meet and fertilize the oocyte, so sperm motility is a requisite for normal fertilization. Asthenozoospermia, or low sperm motility, is a common cause of human male infertility. This is a retrospective study (1992-1999) to document the prevalence of this pathology in infertile men and to clarify the probable factors associated to its etiology. The prevalence was 18.71% for asthenozoospermia and 63.13% for asthenozoospermia associated with oligo- and/or teratozoo-spermia; thus, 81.84% of the studied samples had altered motility. Leukocytospermia, the ratio of germ cells/sperm, anti-sperm antibodies, consistency, biochemical markers of accessory sex glands, and sperm response after swim-up were studied in normospermic (N), asthenozoospermic (A), and combined asthenozoospermic (C) samples. No significant difference was found in the frequency of leukocytospermia among groups. The rate of germ cells/(spermatozoa + germ cells) between C and N (p < .01) and C and A (p < .01) was statistically different, while no difference was found on comparing N and A. MAR-test over 40% was found in 6% of the A samples and 7.6% of the C, while no positive values were observed in the N group. The percentage of hyperviscous samples was higher in the low sperm motility samples than in the normal group. Data on concentration of the biochemical markers seem to be decreased in asthenozoospermia. Pure and combined asthenozoo-spermia showed different behavior in sperm recovery after swim-up. Two different asthenozoospermias could be defined: the pure one where sperm environment is involved (immunological factor, hyperviscosity, and secretory gland function) and the combined, where the testis is comprised.

Epididymal cytology changes in hypothyroid rats.

The purpose of this research was to investigate, using light, transmission, and scanning electron microscope, the effect of hypothyroidism on the ultrastructure of the rat epididymis. Thyroidectomy was obtained by ip injection of 270 microCi of (131)I per rat. One month later, several portions of cauda epididymis were examined. Morphological differences were detected in the epididymis of the hypothyroid animals when compared to the control normal rats. The hypothyroid conditions were associated with important changes in the epididymis. The light observations showed cells with clearing of the chromatin and increased density and thickness of the chromatic rim, chromatinic net, and disappearance of the segment of the chromatin rim. In the scanning electron microscope broken, oblique, denuded epithelial cells with loss of stereocilia were observed, as well as flattening of the tubule. The hypothyroid condition under transmission electron microscope was associated with a decrease in the height of the cells, diminution of the internal lumen and number of mitoses, and decreased chromatin decondensation. Results obtained confirmed that hypothyroidism causes marked structural changes in the ductus epididymis and could adversely affect the maturation and motility of sperm.

Shedding of immature germ cells.

The immature germ cells (IGC) constitute the highest percentage (90%) of nonsperm cells (NSpC) in ejaculates from fertile or infertile men. The objective of this study was to evaluate IGC concentration and the IGC/(IGC + Sp) ratio, in normozoospermia and dispermia. Normozoospermia from men with proven fertility (NPF). nonproven fertility (NNPF). dispermia (D) and semen samples with excessive shedding of immature germ cells (GI 1.7 x 10(6) to 5 x 10(6) IGC/mL and GII > 5.0 x 10(6) IGC/mL) were used in this study. The mean value +2 SD for the NNPF (1.7 x 10(6)/mL) and the value proposed by WHO (5 x 10(6)/mL) were employed to define GI and GII groups. IGC concentration is statistically different in the studied groups. The IGC/Sp ratio showed a significant difference only between the NNPF and the D. When comparing semen parameters (Sp/ejaculate. grade (a) motility and morphology) there was a highly significant difference between NNPF and GI and GII: no difference was found between GI and GII. While studying 200 cases of dispermias 83% showed a high shedding of immature germ cells. The cytological study of nonsperm cells and the count and identification of the immature germ cells could be used to evaluate the dispermic disorders.

Epididymal scanning electron microscopy study in hypothyroid rats.

The purpose of this research was to investigate, using scanning electron microscopy, the effect of hypothyroidism on the ultrastructure of the rat cauda epididymis. Thyroidectomy was obtained by ip injection of 270 muCi of 131I per rat. One month later, several portions of cauda epididymis were examined. Morphological and physiological differences were detected in the cauda epididymis of the hypothyroid animals when compared to the control normal rats. The hypothyroid condition was associated with important changes in the luminal surface of the cauda epididymis epithelium. Broken, oblique, and loss of stereocilia, denuded epithelial cells, and flattening of the tubule were observed. The results confirm that hypothyroidism causes marked structural changes in the cauda ductus epididymis and could be adversely affect sperm maturation motility.

High-affinity binding of T3 to epididymis nuclei.

Thyroid hormones play an important role in epididymal function. Hypothyroid animals experience a significant decrease in the number and forward motility of sperm and a remarkable impairment of epididymal morphology. However, it is yet unknown if such activity is due to direct actions of iodothyronines on the target epididymis. The eventual identification of T3 receptors in the nucleous of epididymal cells becomes relevant. For this reason, the authors searched for specific high-affinity binding of T3 to these nuclei. Twenty prepuberal male Wistar rats were used. The testes and epididymis were approached as one unit through a scrotal incision. The fat-free epididymides were subjected to standard techniques to prepare the nuclei for incubations with 125I-T3 concentrations, ranging from 0.5 x 10(-9) to 2.0 x 10(-11) M. Calculations of association constants and binding capacities were performed according to Scatchard. A single binding site with a Ka of 3.06 +/- 0.6 x 10(9) M(-1) or Kd of 3.26 +/- 0.6 x 10(10) M and a maximal binding capacity of 0.11 +/- 0.02 pmol T3/microg DNA were observed. It is concluded that these nuclei contain a specific T3 receptor. This finding strongly suggests that thyroid hormones have direct effects on the epididymis.

[Usefulness of AgNOR technique in the interpretation of serous effusions]. / Utilidad de la técnica de AgNOR en la interpretación de los derrames de cavidades serosas.

BACKGROUND: AgNOR technique detects, using silver salts, argyrophylic proteins of the nucleolar organizer region (NOR). The number and size of NOR reflect cell activity, proliferation and transformation and may help to differentiate benign from malignant cells. AIM: To assess the value of AgNOR assay to differentiate reactive mesothelial cells from malignant cells in serous effusions. MATERIAL AND METHODS: Thirty one fluids obtained from 16 pleural, 14 peritoneal and one pericardial effusion, were studied. The fluids were processed with Giemsa and Papanicolau stains and with the AgNOR technique. The number of AgNOR dots were counted (only when it was possible to distinguish each individual dot) and the mean value per nucleus was calculated for each smear. RESULTS: Mesothelial cells had a mean of 4.88 +/- 1.5 dots compared with 13.78 +/- 3.88 dots in the malignant cells (p < 0.001). CONCLUSIONS: AgNOR assay can be useful for the differentiation of benign and malignant cells in serous effusions.

Changes in the epididymal ultrastructure in hypothyroid rats.

The purpose of this research was to investigate the effect of hypothyroidism on the ultrastructure of the rat cauda epididymis. Thyroidectomy was obtained by i.p. injection of 270 microCi of 131I per rat. One month later, several portions of cauda epididymis were examined in an electron microscope. Morphological and physiological differences were detected in the cauda epididymis of the hypothyroid animals when compared to the control normal rats. The hypothyroid condition was associated with a decrease in the height of the cells and diminution of the internal lumen, number of mitoses, and chromatin decondensation. Hypothyroidism produces a remarkable impairment of epididymal morphology that could be mainly responsible for the functional alterations that lead to the dramatic slowing of spermatozoa motility, commonly seen in severe thyroid insufficiency.

Thyroid gland and epididymal sperm motility in rats.

The purpose of this research was to investigate the effect of hypofunction of the thyroid gland, caused by radioactive suppression of the gland, on the pattern of spermatozoa motility in different segments of the rat epididymis. Thyroidectomy was obtained by i.p. infection of 270 microCi of I-131. After about 30 days, the animal reached hypothyroidism as determined by serum level of T4. When the motility pattern of the sperm obtained from the epididymis of normal rats was compared to that of hypothyroid animals, a drop in the parameter of path velocity (VAP), progressive velocity (VSL), and track speed (VCL) were detected. Hypofunction was associated with decreasing sperm motility in the epididymis. In thyroidectomized rats injected with T4, no sperm motility changes were observed.

Viscosity of human seminal fluid: role of lysozyme.

The aim of this research was to evaluate the role of lysozyme in the phenomenon of seminal hyper-viscosity. The enzyme was determined in 142 samples of seminal plasma either leucospermic or not, with or without active macrophages classified according to their consistency (normal or high). The kinetic method with Micrococcus lysodeikticus as substrate was employed. No difference was found in enzymatic concentration expressed in nmol/L of enzymatic protein (mean +/- 2 SEM) on comparing normal and high seminal consistency groups, while differences proved highly significant in batches either leucospermic or not (n = 44, 197.2 +/- 51.3 vs. n = 98, 108.3 +/- 12.8; p < .0005). On subdividing the normal and high-consistency groups according to the count of polymorphonuclear leucocytes and the macrophagic responses, differences were also significant (p < .005 in both cases). Lysozyme concentration increases in presence of leucospermic reaction. In vitro lysozyme addition showed no significant effect on samples with high consistency. The results indicate that lysozyme plays no direct role in the phenomenon of seminal hyperviscosity, although its deficiency in cases of chronic infections may prove a factor aggravating the clinical picture.

Phagocytosis of ejaculated spermatozoa.

In patients with accessory gland infections or subjects who have sperm antibodies in their semen, the presence of macrophages with phagocytic activity on ejaculated spermatozoa is significant. Light microscopy cannot certify phagocytosis because it does not give a three-dimensional view of the cells and can lead one to mistake superficial adherence of the spermatozoa to the macrophage for phagocytic activity. For that reason, scanning electron microscopy was used in this study. The samples, fixed with 2.5% glutaraldehyde in phosphate-buffered saline, were processed for observation with light microscopy (Giemsa or Papanicolaou stain) or with scanning electron microscopy (cell selection, critical point drying and paladium-platinum sputtering). With scanning electron microscopy, inactive macrophages had large membrane folds and a globular structure similar to those seen in ascites, whereas when active, they decreased in volume and developed a surface with granules or blebs. Inactive macrophages were rarely seen. A few minutes after mixing the different fractions of the ejaculate, phagocytosis reached such a level of activity that the spermatozoa partly covered the macrophages. Thus, we observed that the spermatozoa were caught by the head first in some instances but by the main-piece fragment of the tail first in other instances; very rarely were they taken by the midportion, between the head and tail. The presence in the ejaculate of macrophages with phagocytic activity on living, motile spermatozoa thus indicates that the encounter between the macrophages and spermatozoa was a result of the assemblage of components that make up the ejaculate. In this way the contributions of the prostatic gland and seminal vesicles play an important part in the spermiophagy of spermatozoa.

'Ciliated' tumour cells in ascitic fluid from two cases of cystadenocarcinoma of the ovary.

We report two cases of primary carcinoma of the ovary in which 'ciliated' adenocarcinoma cells were found in the ascitic fluid. Transmission electron microscopy revealed that these were not true cilia but rather a prolific growth of abnormal microvilli. The cytological findings were compared with the histological appearances of the primary tumour. No ciliated cells were seen in the primary tumour, suggesting that the formation of the microvilli represented an independent proliferation of the cells in the fluid. Special staining reactions for mucin, alkaline phosphatase and epithelial membrane antigen were identical in the primary tumour and the cells in the ascitic fluid.

Citología del líquido amníotico / Cytology of the amniotic fluid

En el líquido amniótico es de uso generalizado la determinación del porcentaje de células naranjas en los estudios de madurez fetal. Puede accederse a otros datos de valor, mediante el uso simultáneo a la coloración con sulfato de azul Nilo, de coloraciones morfológicas tales como la de Papanicolaou. Esta brinda la posibilidad de realizar la determinación de los porcentajes de células anucleadas y amnióticas, del índice picnótico y además posibilita el reconocimiento de los distintos tipos celulares, en los casos en los que resulte necesario. Agrupando las gestas en períodos que abarcan: menos de 34 semanas de 34 a 37 semanas, de 38 a 40 semanas, los datos obtenidos son: a) Porcentaje de células anucleadas: 3,1 ñ 2,2; 10,9 ñ 7,0; 21,1 ñ 17,1; 22,7 ñ 16,8. b) Porcentaje de células amnióticas: 2,2 ñ 1,8; 1,1 ñ 1,0; 0;0. c) Recuento celular: 57,6 ñ 54,1; 69,7 ñ 49,7; 443,2 84,1; 487,9 93,3 respectivamente. La limitación de las determinaciones citológicas reside en el alto desvío standard que presentan. Por ello el empleo simultáneo de varias, permite aumentar la exactitud de los resultados

Citología del líquido amníotico / Cytology of the amniotic fluid

En el líquido amniótico es de uso generalizado la determinación del porcentaje de células naranjas en los estudios de madurez fetal. Puede accederse a otros datos de valor, mediante el uso simultáneo a la coloración con sulfato de azul Nilo, de coloraciones morfológicas tales como la de Papanicolaou. Esta brinda la posibilidad de realizar la determinación de los porcentajes de células anucleadas y amnióticas, del índice picnótico y además posibilita el reconocimiento de los distintos tipos celulares, en los casos en los que resulte necesario. Agrupando las gestas en períodos que abarcan: menos de 34 semanas de 34 a 37 semanas, de 38 a 40 semanas, los datos obtenidos son: a) Porcentaje de células anucleadas: 3,1 ñ 2,2; 10,9 ñ 7,0; 21,1 ñ 17,1; 22,7 ñ 16,8. b) Porcentaje de células amnióticas: 2,2 ñ 1,8; 1,1 ñ 1,0; 0;0. c) Recuento celular: 57,6 ñ 54,1; 69,7 ñ 49,7; 443,2 84,1; 487,9 93,3 respectivamente. La limitación de las determinaciones citológicas reside en el alto desvío standard que presentan. Por ello el empleo simultáneo de varias, permite aumentar la exactitud de los resultados (AU)