Search

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.

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.

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.

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.

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.