Hypogonadotrophic hypogonadism (HH) is characterized by deficient gonadotrophin secretion, resulting from pituitary or hypothalamic defects. In order to induce spermatogenesis, HH patients are treated with commercially available gonadotrophins. As far as is known, quality and genetic integrity of induced sperm cells have never been investigated, although they represent an important issue, since the ultimate goal of this treatment is to have competent spermatozoa in order to achieve paternity. In order to evaluate the nuclear integrity of induced sperm cells, sperm samples from treated HH patients were compared with sperm samples from normospermic control donors. Sperm cells were analysed by fluorescence in-situ hybridization, using probes specific for chromosomes 13, 21, 18, X and Y, and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Results showed that the rate of aneuploid and diploid sperm cells in patients was not statistically different from controls and that the rate of sperm cells with fragmented DNA was within the normal values. Spermatozoa obtained by gonadotrophin treatment in HH patients are likely to have a balanced chromosomal content and a normal DNA integrity but this conclusion needs to be confirmed by further studies dealing with a greater number of patients.Hypogonadotrophic hypogonadism (HH) is characterized by deficient gonadotrophin secretion, resulting from pituitary or hypothalamic defects. In order to induce spermatogenensis, HH patients are treated with commercially available gonadotrophins. As far as is known, quality and genetic integrity of induced sperm cells have never been investigated, although they represent an important issue, since the ultimate goal of this treatment is to have competent spermatozoa in order to achieve a paternity. In order to evaluate the nuclear integrity of induced sperm cells, sperm samples from treated HH patients were compared to sperm samples from normospermic control donors. Sperm cells were analysed by FISH, using probes specific for chromosomes 13, 21, 18, X and Y, and by TUNEL assay. Results showed that the rate of aneuploid and diploid sperm cells in patients was not statistically different from controls and that the rate of sperm cells with fragmented DNA was within the normal values. Spermatozoa obtained by gonadotrophin treatment in HH patients are likely to have a balanced chromosomal content and a normal DNA integrity but this conclusion needs to be confirmed by further studies dealing with a greater number of patients.

We quantified all fetal nucleated cells (FNCs) per unit volume of maternal blood in different aneuploid pregnancies using molecular cytogenetic techniques. Seven cases of male trisomy 18, two triploidies (69,XXX), two 47,XXX, one 47,XXY, one 47,XYY, one male trisomy 13, and one case of 47,XY,r(22),+r(22) were analyzed. Whole blood samples were obtained from 15 women between 17 and 29 gestational weeks and harvested without using fetal cell enrichment procedures. Fluorescence in situ hybridization and primed in situ labeling were performed to identify the FNCs. All slides were manually scanned to quantify those cells. We have identified 4–20 FNCs/ml of maternal blood in the cases of trisomy 18; 10 and 25 FNCs/ml in the two cases of triploidy; 16 and 14 FNCs/ml, respectively, in the two X trisomies; 19 FNCs/ml in the 47,XXY; 26 FNCs/ml in the 47,XYY; nine FNCs/ml in the trisomy 13; and 10 FNCs/ml in the case of r(22). To detect all FNCs in all aneuploid pregnancies, we have used a very simple method that minimizes the manipulation steps to avoid losing fetal cells. The number of FNCs identified in aneuploid pregnancies was 2–5 times higher than in normal pregnancies. This higher number of FNCs will favor the design of a non‐invasive pre‐natal test.

Background Prenatal diagnosis of trisomy 21 is based on fetal karyotyping generally obtained using invasive methods. During pregnancy, the circulating fetal cells in maternal blood constitute a potential source for development of a noninvasive prenatal diagnosis. The objective of this study was the identification and quantification of all fetal nucleated cells per unit volume of peripheral blood of pregnant women carrying male fetuses with trisomy 21 using molecular cytogenetic techniques.Methods Peripheral blood samples were obtained from 16 women carrying male fetuses with trisomy 21. We used a simple and rapid method of harvesting blood without recourse to any enrichment procedures or cell-separation techniques. To evaluate the potential of this method, 16 specimens were analyzed by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) using specific probes to chromosomes X, Y and 21.Results The number of fetal cells varied between 6 and 32 per mL of maternal blood. This number is 3-5 times higher than that from normal pregnancies.Conclusions Our current results are in agreement with the results previously reported by other groups showing that the number of fetal cells in maternal blood in trisomic 21 pregnancies is higher than in normal pregnancies. This high number of fetal cells is regarded as an advantage for the development of a noninvasive prenatal diagnostic test. Copyright (c) 2006 John Wiley & Sons, Ltd.

The aim of this study is to validate the multi-PRINS (primed in situ labelling) technique for simultaneous detection of chromosomes 18, X and Y in uncultured amniocytes for prenatal diagnosis of aneuploidy. The sites of the newly synthesized DNA sequences were showed as fluorescent signals by using immunochemistry. A multi-PRINS technique was specifically performed for simultaneous detection in the same cells of three chromosome targets, e.g. chromosomes 18, X and Y. Fluorescent signals corresponding to chromosomes 18, X and Y were showed as yellow, red and green colour spots, respectively. A multi-FISH technique using chromosome 18, X and Y probes was performed for comparison. Sixty cases were analysed using both multi-PRINS and multi-FISH. Fifty to two hundred nuclei were scored for each case for each technique. In all cases, there was no significant difference in the detection of chromosomes 18, X and Y regarding the sensitivity, the specificity and the efficiency; multi-PRINS and multi-FISH yield a similar distribution of the number of spots per nucleus. Both techniques were able to identify aneuploid cases without any ambiguity. Both multi-PRINS and multi-FISH can accurately and reliably detect aneuploidies involving chromosomes 18, X and Y in uncultured amniocytes. Finally, multi-PRINS represents a faster and more cost-effective alternative to FISH for prenatal testing of aneuploidy in uncultured amniocytes.

As a non-isotopic molecular cytogenetic technique, the primed in situ (PRINS) labelling reaction represents a major technological progress achieved in the past decade. It has become a routine technique for the microscopic visualization of specific DNA sequences in cells and nuclei and constitutes a good alternative to the fluorescence in situ hybridization (FISH) procedure. Among the multiple advantages that characterize the PRINS technique, specificity, rapidity, reliability, reproducibility, and cost-effectiveness can be mentioned. PRINS can be in addition associated with other techniques like FISH, indirect immunofluorescence, and nick translation. The most recent developments show the great potential of this technique. Now PRINS can be used to study single-copy genes and, consequently, can be routinely used to investigate deletions associated with microdeletion syndromes. Therefore, the PRINS technique has the potential to become a widely used molecular cytogenetic tool in clinics and research. This short review presents how the PRINS technique contributed to further the understanding of biological phenomena and describes the different possibilities and applications of the PRINS method in several biological and clinical fields (pre-implantation testing, prenatal, constitutional and oncologic genetic diagnosis).

Le diagnostic positif et étiologique d’une alopécie chez l’enfant est important pour différencier les causes bénignes (également les plus fréquentes) pouvant prétendre à un traitement et ne devant pas inquiéter les parents à tort, des alopécies qui peuvent être le reflet d’une cause interne ou le premier signe clinique orientant vers une maladie syndromique. Nous proposons une classification en différenciant les alopécies de survenue congénitale ou précoce qui sont spécifiques de la période infantile, et les alopécies plus tardives survenant au cours de l’enfance ou de l’adolescence que l’on peut également retrouver chez l’adulte. Les signes dermoscopiques et histologiques des pathologies seront évoqués, ainsi que les anomalies cutanées ou atteintes d’autres organes permettant de poser le diagnostic.

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 10 4 to 1 per 10 9 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non‐invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.

Four new cases of holoprosencephaly are described in fetuses exhibiting abnormal karyotypes with different distal and proximal rearrangements of the long arm of chromosome 7. Three of them showed terminal deletions of chromosome 7q, confirming the importance of the 7q36 region in holoprosencephaly. The karyotype of the fourth fetus showed an apparently balanced de novo translocation, t(7;13) (q21.2;q33), without any visible loss of the distal part of chromosome 7q. The involvement of new genes, different from the human Sonic Hedgehog gene (hShh) responsible for holoprosencephaly, or a positional effect are discussed.

A patient with mixed gonadal dysgenesis and Y isochromosomes i(Y) is described. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and several other cell lines with two different marker chromosomes (mars). These markers had either a monocentric (mar1) or a dicentric appearance (mar2). Following high-resolution GTG, RBG, QFQ, and CBG bandings, five cell lines were identified; 45,X/46,X, + mar1/46,X, + mar2/47,X, + mar1x2/47,X, + mar2x2. The percentages were 66/6/26/1/1%, respectively. Chromosome banding analyses were insufficient for characterization of the markers. In situ hybridization of specific probes for the Y centromere and its short arm showed, both in fluorescence and electron microscopy (EM), two different Y rearrangements. Mar1 is an isochromosome for the short arm i(Yp) and mar2 is a dicentric which was shown by EM to be a double isochromosome Yp, inv dup i(Yp). The breakpoint producing mar1 is within the centromere and the one producing mar2 is within one of the short arms of the Y isochromosome. The findings of different cell populations in peripheral blood lymphocytes indicate the postzygotic instability of this i(Yp). © 1995 Wiley-Liss, Inc.