Identification of two human sperm populations using flow and image cytometry.

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder ("marginal population"). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide.

Comparison of the chromatin stainability of human spermatozoa separated by discontinuous Percoll gradient centrifugation. A flow cytometric contribution.

Many clinical programs in in vitro fertilization (IVF) use the classic Percoll sperm preparation technique to obtain a subpopulation of highly mobile spermatozoa. This Percoll discontinuous gradient centrifugation technique (densities ranging from 1.042 to 1.116 g/mL) was used at room temperature to separate human spermatozoa fractions in order to determine differences in chromatin stainability. The stainability was measured by analysis of DNA fluorochrome uptake by flow cytometry using human peripheral blood lymphocytes as an internal standard. A significant difference in chromatin stainability was noted between the residual fraction (immobile spermatozoa remaining at the top of the Percoll gradient) (44.2%) and the selected one (highly mobile spermatozoa harvested in the 90% and 100% steps) (36.3%). The sperm DNA heterogeneity, as defined by the coefficient of variation (CV), was significantly lower for the selected fraction as compared to the residual one (CV = 12.8% versus 15.4%). A marginal population of spermatozoa adjacent to the germinal peak was also identified. The percentage of this marginal population was significantly lower for the selected fraction as compared to the residual one (7.4% versus 22%). Using a biochemical in vitro decondensation method, the chromatin stainability of the selected and residual fractions (56.9% versus 62.9%) was also determined, demonstrating that the chromatin of the selected fraction was less stainable. This study confirmed that Percoll centrifugation selects a germinal population that is denser and less stainable than with other techniques and analyzed the relation between density, high mobility and probable capacitation.

Quantitative assessment of chromatin stability alteration in human spermatozoa induced by freezing and thawing. A flow cytometric study.

The authors examined the effect of cryopreservation on chromatin stability in human spermatozoa from 21 ejaculates. Each ejaculate was divided into four aliquots: (1) fresh aliquot, (2) frozen and thawed aliquot, (3) fresh aliquot subjected to an in vitro decondensation method, and (4) frozen and thawed aliquot subjected to the same in vitro decondensation method; all were then fixed with an ethanol fixative agent. Chromatin stainability was quantified by flow cytometric measurement of fluorochrome uptake by DNA. Study of 21 fresh aliquots showed that 37.9% of the DNA was accessible to propidium iodide. The comparative stainability between the 21 fresh and 21 frozen-thawed, undecondensed aliquots demonstrated a low but significant increase in accessibility of DNA by propidium iodide for the thawed samples: 38.7 +/- 1.7% (mean +/- SD) versus 37.9 +/- 1.3%. The biochemical action of the nuclear decondensation solution increased the accessibility of propidium iodide, but in different ways: 57.2 +/- 12.9% versus 54.7 +/- 13.7%, respectively, for fresh and frozen-thawed aliquots. Analysis of the flow cytometric histograms revealed an intermediate population of spermatozoa adjacent to the main germinal peak. This population increased significantly: 9.6 +/- 1.9% for the fresh versus 12.3 +/- 4.9% for the frozen-thawed undecondensed aliquots and 8.6 +/- 3.5% versus 12.3 +/- 4.9%, respectively, for fresh and frozen-thawed, decondensed aliquots. Because the chromatin stability of thawed spermatozoa may be a critical factor in assisted procreation, the authors discuss the effect of thermal denaturation on the nucleoprotein structures and the origin of the intermediate population of spermatozoa.

Flow cytometric analysis of DNA content in colorectal polyps resected at endoscopy.

Samples from 64 consecutive resected colorectal polyps were preserved in liquid nitrogen and analyzed by flow cytometry to assess the nuclear DNA content, which was compared to the histopathologic findings. The frequency of aneuploidy in these nonselected polyps was 17.2%. There was a significant correlation between the diameter of the polyp and the frequency of aneuploidy (P = .04), with all aneuploid polyps being greater than or equal to 10 mm. Similarly, aneuploidy was significantly more frequent for polyps that were both greater than or equal 20 mm and showed at least severe dysplasia (P = .02). On the other hand, there was no correlation between the ploidy and the gross histopathologic type (tubular, tubulovillous or villous), and the proliferation index did not correlate with any of the parameters studied.

Quantitative assessment of human spermatozoa chromatin stainability by flow cytometry: preliminary study.

The authors report a human sperm suspension method to assess quantitatively the stainability of the spermatozoa chromatin. Analysis of 15 ejaculates demonstrated the existence of a constant percentage of stained DNA in different ejaculated spermatozoa. The condensed chromatin stainability was then compared to the in vitro decondensed chromatin stainability with the finding of an increased uptake of fluorochrome in relation with the nuclear chromatin decondensation. The quantitative spermatozoa chromatin stainability and its decondensation aptitude may be of importance in sperm physiology.

Automated image analysis of in vitro decondensation of human spermatozoa nuclei. III. Variable decondensation with and without incubation in the seminal fluid.

The effect of seminal fluid upon human spermatozoa was analyzed using in vitro chromatin decondensation and automated image analysis. A number of specimen portions processed after incubation in seminal fluid showed different total mean areas as compared to the corresponding portions processed immediately. Comparison of the results obtained with and without delay showed that the incubation in seminal fluid promoted decondensation in some cases, but retarded it in others. Thus, the seminal fluid stabilized the chromatin condensation in some spermatozoa, but not all. The stabilization may be due to the influence of prostatic zinc.

Bronchial carcinoma and image analysis. Differentiation between poorly differentiated epidermoid carcinoma and small-cell carcinoma.

The utility of automated image analysis in the distinction between poorly differentiated epidermoid carcinoma (eight cases) and small-cell carcinoma (ten cases) was studied. Material obtained using the bronchial brushing technique was prepared by a cytocentrifugation technique. In each case, a total of 100 bronchial cell nuclei were selected using the Leitz TAS, which measured eight parameters per cell in order to ascertain the homogeneity or the heterogeneity of the nuclear populations. Except for one sample exhibiting preparation artifacts, the method proved capable of differentiating between these two types of bronchial carcinoma, with heterogeneity of the malignant nuclei indicating an epidermoid carcinoma and homogeneity indicating a small-cell carcinoma. A correlation was observed to exist between the morphologic and the morphometric criteria.

In vitro decondensation of the human spermatozoan nucleus and image analysis. Quantitative data on the kinetics of in vitro decondensation of the human spermatozoan nucleus.

The authors subjected 14 human sperm samples to decondensation in vitro. They analyzed the images of a total of 9,681 objects as a function of time of decondensation. The discussion bears on the heterogeneity of the process of chromatin decondensation in human spermatozoa, on the influence of the zinc/fructose ratio in the seminal fluid and on the role of the period during which the spermatozoa are left in contact with the fluid.

Automated cytopathologic diagnosis of bronchial carcinoma. II. Preliminary results of classifying cytocentrifuged bronchial brushings.

An automated discrimination between healthy and neoplastic bronchial cells was performed on eight bronchial smears prepared by cytocentrifugation. An image analyzer was used to examine 415 cells in these smears. The nuclear surface of each cell was measured, as was the total integrated optical density for 25 programmed thresholds. The results show that it is possible to distinguish healthy from cancerous cells in a given subject using these two measured parameters and two new parameters deduced mathematically. It appears difficult, however, to demonstrate a typical healthy and typical cancerous bronchial cell that could be used as a reference for all subjects. It is thus the presence of cell heterogeneity in a given subject that enables him or her to be characterized as healthy or having cancer.

[In vitro decondensation of the human spermatozoan nucleus and image analysis]. / Décondensation in vitro du noyau du spermatozoïde humain et analyse d'image.

Between the first stage: differentiation of the human spermatozoa from spermatid in testicle, and the second one: formation of male pronucleus in ovum, the nucleus of spermatozoa undergoes a chromatin condensation and a chromatin decondensation mechanism. Besides it is possible to get this decondensation mechanism in vitro by putting spermatozoa into specific solution with the purpose of carry out an image analysis. The aim of this study was to manage a quantitative analysis of the in vitro nuclear chromatin decondensation of human spermatozoa with an image analyzer of T.A.S. "texture analysis system".

Automatic cytopathologic diagnosis of bronchial carcinoma. I. Cytocentrifugation of bronchial brushings for image analysis.

A cytocentrifugation technique for the preparation of monodispersed preparations of bronchial cells collected by brushing is described. First developed with the aim of obtaining slides suitable for automated analysis, it was found to be useful for routine morphologic examinations. This technique requires careful preparation as well as "education" of the pathologist/screener in the appearance of the cellular elements in its samples. However, it is reliable and reproducible and, above all, eliminates a certain number of artifacts found in conventional preparations.

[Cytology in the detection and followup of urothelial tumors. Critical review of 500 cytologic investigations (authors transl)]. / La cytologie urinaire dans le dépistage et la surveillance des tumeurs urothéliales. Résultats à propos de 500 bilans.

500 cytologic studies were performed in patients who underwent urological explorations. Sensitivity is defined as the ability to identify correctly those patients who have tumoral disease of urothelium: urine cytology is ineffective in detecting benign papilloma, but sensitivity is more than 66% in low grade carcinoma and 80% in high grade carcinoma; cytology was able to detect 4/6 transitional cell carcinomas of the upper urinary tract. Specificity, which is defined as the ability to identify correctly those patients who do not have tumoral disease, is extremely high; thus, careful urological examination must be completed when cytology is positive. Urinary cytology is needed in every urological exploration concerned with urinary tract.

[Nucleo-cytoplasm ratio, DNA content of superficial and intermediate vaginal cells during the female genital cycle]. / Rapport nucléo-cytoplasmique, contenu en DNA des cellules vaginales superficielles et intermédiaires au cours du cycle génital, féminin.

A quantimetric study of vaginal superficial cells is done every day of the sexual cycle on five normal women (no pill) with a cervical smear of type 2 (Papanicolaou classification). Our results show that nucelo-cytoplasmic ratio for 98% of the cells varies between 0.0040 and 0.0210. This level is independant of age and cycle duration and is proposed as reference for later studies of women under pill or with cervico-vaginal dysplasia and cancer.