[A simple, low-cost and non-invasive method for screening Y microdeletions in infertile men]. / Evaluation d'une technique simple et rapide de détection de microdélétions du bras long du chromosome Y.

OBJECTIVES: Recent investigations showed a high prevalence of Y chromosome microdeletions in men with severely impaired spermatogenesis. Screening for these men is recommended prior to assisted reproduction techniques. The aim of this study was to set up a simple method to detect Y deletion in infertile men. First, we tested the feasibility of cytobrush to collect oral cells as source of DNA. Second, we compared a classic PCR corresponding to European recommendations to the Promega kit. PATIENTS AND METHODS: Seventeen infertile male patients with previously characterized deletions were included in the present study, after fully informed written consent. Both oral cells and blood were used for DNA extraction. A specific DNA extraction protocol was carried out on the buccal cells. The DNAs were tested for Y deletion screening by two different methods. RESULTS: We retrieved between 4 and 10 microg of DNA per brush from buccal cells, allowing several multiplex PCR. The Promega kit detected all the deletions but one: an AZFa deletion was not detected by the two markers of the kit covering this region. In addition, sY130, sY133 and SY153, included in the kit, are not reliable. DISCUSSION AND CONCLUSIONS: Buccal cells represent a convenient substitute for blood in testing for Y microdeletions. Both false negative and false positive results were obtained with Promega Kit. On the opposite, PCR according to the European recommendations allow the accurate detection of Y microdeletion in our 17 cases, at a lower cost.

[Evaluation of the interest of semen culture before cryoconservation]. / Evaluation de l'intérêt de la spermoculture avant autoconservation de sperme.

OBJECTIVES: French guidelines recommend screening all patients for virus infection prior to cryopreservation of their semen. In case of viral risk, the use of specific high secure CBS straws is recommended. The objective of this work was to evaluate the microbiological risk by testing all semen samples before cryopreservation. PATIENTS AND METHODS: Fifty one patients underwent a semen culture before cryopreservation. RESULTS: The fifty one patients were classed into 3 groups following semen culture results: group I: negative culture (39/51, 76.47%), group II: positive culture with microbiological contamination (7/51, 13.73%) and group III: positive culture with pathogen (5/51, 9.8%). For 3 patients of the latter group, we tested a three-layer density gradient to eliminate bacteria before Assisted Reproductive Techniques. DISCUSSION AND CONCLUSIONS: This paper discusses the risks related to microbiological contamination and the options available in this case.

A simple, low cost and non-invasive method for screening Y-chromosome microdeletions in infertile men.

BACKGROUND: Recent investigations emphasized a high prevalence of Y-chromosome microdeletions in men having severely impaired spermatogenesis. Screening of these men is recommended prior to assisted reproduction techniques. METHODS: The aim of this study was to define a reliable and efficient method to detect Y-chromosome deletions in infertile men. At first the feasibility of using a cytobrush to collect buccal cells as a source of DNA was tested. Then, a multiplex PCR in accordance with European recommendations (European Andrology Academia: EAA) was compared with a commercial kit. The test population consisted of 18 infertile male patients (with a known Y-deletion). Both buccal and blood cells were used for DNA extraction. A specific DNA extraction protocol was carried out on the buccal cells. RESULTS: Between 4-10 micro g of DNA were retrieved per brush, allowing for several PCR attempts. The commercial kit failed to detect an AZFa deletion. Furthermore, markers sY130, sY133 and sY153, included in the kit, are not reliable. Both false negative and false positive results were generated by the commercial kit. CONCLUSION: A multiplex PCR performed pursuant to EAA recommendations is proposed. When the testing is conducted with DNA extracted from buccal cells, this protocol is simple, accurate and affordable.

Pregnancy after safe IVF with hepatitis C virus RNA-positive sperm.

In France, assisted reproductive technology (ART) for hepatitis C virus (HCV)-infected patients is now subject to strict control after the publication of recent guidelines. Infertile serodiscordant couples (HCV-viraemic men and their seronegative female partners) require special care to carried out in designated 'viral risk' laboratories. Twelve sequential semen samples taken from an HCV chronically infected patient were analysed within 22 months. HCV RNA was detected in all the seminal plasma sampled before antiviral treatment with relatively high viral loads, and in two of the corresponding fractions of motile sperm obtained after a gradient selection, suggesting that a contamination risk by HCV through ART cannot be excluded. When the selection of sperm on a discontinuous gradient was followed by an additional swim-up step, HCV RNA was never detected in the motile sperm suspension that was frozen in highly secure straws. IVF was performed using cryopreserved sperm that tested negative for HCV RNA, resulting in a pregnancy. One month after embryo transfer, testing for HCV RNA and antibodies in the woman gave negative results.

Prospective flow cytometric DNA analysis of hepatocellular carcinoma specimens collected by ultrasound-guided fine needle aspiration.

BACKGROUND: The survival of 52 patients with hepatocellular carcinoma (HCC) seen during the last 4 years was analyzed prospectively on the basis of disease stage and nuclear DNA content. METHODS: Ploidy was measured by flow cytometry (FCM). Cells for cytologic diagnosis and FCM were collected by ultrasound-guided fine needle aspiration. RESULTS: DNA aneuploidy, which was detected in 62% of the patients, did not correlate with clinicopathologic features, except in the sonographic aspect (P = 0.03). However, ploidy correlated significantly with survival; the survival times for patients with an aneuploid DNA index were significantly shorter than for those with a diploid index (P = 0.02). In a Cox multivariate analysis, DNA content was prognostically significant, as were the grade of cirrhosis severity and the echographic aspect. CONCLUSIONS: In addition to the clinicopathologic features observed, FCM DNA analysis of ultrasound-guided fine needle aspirates from HCC is a simple and valid method for estimating a prognosis of these patients.

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.

[Impregnation and liberation of nicotine with hydrogen copolymer discs]. / Imprégnation et libération de nicotine à partir de disques de copolymère hydrogel réticulé.

Reticulated copolymers with products N-vinyl-2 pyrrolidone (NVP), hydroxy-2 ethyl (HEMA) and methyl methacrylate (MMA) were realized in disc form. Nicotine was used as active principle. On these discs the following study was carried out: permeation, percentage of liquid absorbed, texture, as well as nicotine release kinetics. A 2-variable (NVP and HEMA/MMA), 5-level experimental plan has enabled the outlining of the significant differences on the action of these parameters. The release kinetics show particularly that these discs behave as forms of prolonged released and that the released quantities are sufficient to envisage the use of these reticulated copolymers within the framework of a transdermal system.

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.

[Value of the peroperative association of echography, extemporaneous cytology and immediately available insulin blood levels of the portal vein. Apropos of 2 cases of insulinoma]. / Intérêt de l'association per-opératoire de l'échographie, de la cytologie extemporanée et des dosages d'insulinémie portale avec résultats immédiats. A propos de deux cas d'insulinome.

The authors report two cases of insulinoma managed by per-operative ultrasound and cytology, together with immediate portal vein insulin levels. They stress the value of these per-operative exploratory techniques which reduce the number of pre-operative invasive examinations while ensuring a greater efficacy in the localization and treatment of these tumors.

[False positive urinary cytology. Diagnostic and prognostic value in the detection and surveillance of urothelial tumors. Apropos of 57 false positive cases in a series of 1000 cytologies]. / Les cytologies urinaires "faussement positives". Valeur diagnostique et pronostique dans le dépistage et la surveillance des tumeurs urothéliales. A propos de 57 cas de "faux positif" sur une série de 1,000 cytologies.

Out of 1,000 optical cytological urine tests carried out over a period of two years, 84 isolated cases were "positive" and alerted 57 patients. These cases are analyzed by the authors with a follow-up period of 48 to 72 months: among those patients already under supervision for urothelial tumours, 40.4% showed urothelial recurrence of which 70%, that did not appear to carry a worse prognosis, were detected in the first twelve months. Since these patients usually underwent annual endoscopic examination, this meant that for 20% (i.e. 4.15% of the group under supervision) the diagnosis of recurrence could be made at least 6 months earlier. This alone is a justification for cytological examination which does not involve any complications but merely entails increased cystoscopic and biopsy tests. On the other hand, there appear to be no grounds to advocate mass "screening" except in populations with warning symptomatology (especially haematuria) for which the ratio of positive findings in this series was 2.3/1,000. The future development of automatic apparatus for cytological tests should obviate the significant personal factor involved in cytological examinations, as shown by a review of the literature.

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.