[Assisted reproductive techniques in women aged 38 years or more]. / Prise en charge en Assistance médicale à la procréation des femmes de 38 ans et plus: résultats d'une enquête à propos de 84 couples.

OBJECTIVE: More and more young women are delaying childbearing until the fourth decade of life: thus, Assisted Reproductive Techniques centres receive more and more requests from ageing women. The aim of the study is to analyse the purpose of these requests, the biological and clinical features of these patients and the results in our infertility centre. PATIENTS AND METHODS: A retrospective study was carried out at the CHU of Saint-Etienne from 01.01.01 to 31.12.04. We analysed the social, clinical and biological features of 84 couples when the woman's age was equal or superior to 38 years, representing 218 cycles. A questionnaire was used to collect social data. RESULTS: Several factors can explain the increasing number of ageing women consulting for infertility: extend university time and professional career, professional stability, contraception and late meeting of the partner, false reassuring information concerning progress in ART, second child desire after a late first pregnancy, but also second marital unions and child desire in the redefined couple. In our study, above 40 years old, the pregnancy (19.4 versus 10.5%) and delivery rates (16.7 versus 5.8%) clearly decreased in IUI. Thus, most of the clinicians propose, in first choice, an IVF cycle to a 40 year-old woman. The ultrasound measurement of antral follicle count can accurately evaluate the prognosis in terms of pregnancy (P<0.01) and delivery rate (P=0.03). For patients with unfavourable prognosis, oocyte donation, embryo donation, or adoption can be considered. DISCUSSION AND CONCLUSION: ART cannot compensate for the natural decrease in pregnancy rates and the increase in early miscarriages in ageing women. Therefore, it is essential to inform young women of the negative effects of age on their potential fertility.

Bacterial risk and sperm cryopreservation.

Prior to sperm cryopreservation, French guidelines only recommend viral screening for serological status towards human immunodeficiency virus, hepatitis B and C viruses and Treponema palidum. The probability of semen infection by other bacterial pathogens is not taken into consideration by the current recommendations. The objective of the present study was to evaluate this risk and a strategy to reduce it prospectively. Ninety-six patients consulting for sperm cryopreservation underwent a semen culture simultaneously to cryopreservation. The patients were classified into three groups following semen culture results: negative culture (group 1, 77/96, 80.2%), positive culture with saprophytic agents (group 2, 9/96, 9.4%) and positive culture with pathogen agents (group 3, 10/96, 10.4%). For six patients of the latter group showing a genital infection with Ureaplasma urealyticum, a discontinuous gradient selection performed on the cryopreserved sample was efficient to discard bacteria. These data emphasize the usefulness to cultivate semen simultaneously to cryopreservation and demonstrate the ability to remove some microbial agents from semen before its use in assisted reproductive techniques.

[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.

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.

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.