Double Density Gradient Centrifugation Research Articles

P-986: Evaluation of seminal fractions using the Toluidine blue test and sperm chromatin structure assay

Over the past decade, several techniques have emerged as potential protocols for evaluating the sperm chromatin integrity. Such integrity is of utmost importance for successful fertilization. Most of the techniques used for sperm chromatin evaluation require extensive instrumentation and special expertise. Toluidine blue (TB) test has been reported as an easily accessible alternative test for sperm chromatin assessment. Spermatozoa may either stain as dark violet (DV, abnormal chromatin conformation/DNA integrity) or light blue (LB, normal chromatin conformation). The sperm chromatin structure assay (SCSA) is the one of the most standardized methods for assessment of fragmented sperm DNA and the percentage of sperm with immature nuclear development (high DNA stainability index, %HDS). The objective of our study was to evaluate the Toluidine blue test and SCSA for assessment of sperm DNA damage in different seminal fraction. Prospective-controlled study. Semen samples were collected from 19 healthy donors. The neat semen samples were divided into mature and immature sperm fractions using double density gradient centrifugation (90% and 47% layers). Both fractions as well as an aliquot of the neat sample were evaluated using the Toluidine blue test. In addition, the %HDS was measured using the SCSA. The means ± standard errors of the percentages of sperm with normal (light blue) and abnormal (dark violet) chromatin in neat semen, mature and immature fractions are given in the table below. Using the Toluidine blue test, mature sperm fractions showed higher incidence of spermatozoa with normal DNA compared to the immature fractions and neat semen samples (p=0.04 and p=0.006, respectively). Similarly, the mature sperm fractions showed lower incidence of spermatozoa with abnormal DNA compared to the immature fractions and neat semen samples (p=0.01 and p<0.0001, respectively). The %HDS showed a significant positive correlation with the number of DV sperm (r=0.51, p=0.0002) and a significant negative correlation with the number LB sperm (r=-0.59, p<0.0001).Tabled 1 Mature sperm fractions show a lower incidence of abnormal DNA as measured by the Toluidine blue test. The association of %HDS with cells that are stained dark violet suggests that Toluidine blue can identify immature nuclear chromatin with abnormal chromatin conformation.

Sperm Recovery Evaluation Following Magnetic Cell Sorting

Double density gradient centrifugation (DGC) is being recognized as a method of choice for sperm preparation for assisted reproduction techniques. However, the sperm density in DGC does not correlate with apoptosis, which may be a cause of fertilization failure. Magnetic cell sorting (MACS) using annexin V-conjugated microbeads effectively separate apoptotic and non-apoptotic spermatozoa based on the externalization of phosphatidylserine (PS) to the outer membrane leaflet. MACS in combination with DGC can be used as a sperm preparation technique to enhance the sperm quality and function.

Detection of fetal trisomy 18 by short-term culture of maternal peripheral blood

Objective: We report the prenatal detection by fluorescence in situ hybridization analysis of a male fetus with trisomy 18. Study Design: Total nucleated cells recovered from 7 mL of maternal peripheral blood by means of double-density gradient centrifugation were cultured for 3 days in a devised medium. Results: Fetal cells with X- and Y-specific signals were detected in all the established cultures, but the yield and purity were higher in the culture from the 1077 Ficoll layer. Cumulatively, 84 fetal cells were recorded by analysis of 5640 cells. The hematopoietic lineages involved in the production of the fetal cells in culture were not assessed. For the cultures established with the 1119 Ficoll layer, the involvement of progenitors or precursors of the erythroid lineage was assumed because postculture sorting was directed toward cells expressing the erythropoietin receptor. Conclusion: We conclude that culturing total nucleated cells from maternal blood is a new procedure that could prove valuable in the detection of the main fetal aneuploidies affecting pregnant populations. (Am J Obstet Gynecol 2000;183:222-5.)

Detection of fetal trisomy 18 by short-term culture of maternal peripheral blood

<h2>Abstract</h2> <b>Objective:</b> We report the prenatal detection by fluorescence in situ hybridization analysis of a male fetus with trisomy 18. <b>Study Design:</b> Total nucleated cells recovered from 7 mL of maternal peripheral blood by means of double-density gradient centrifugation were cultured for 3 days in a devised medium. <b>Results:</b> Fetal cells with X- and Y-specific signals were detected in all the established cultures, but the yield and purity were higher in the culture from the 1077 Ficoll layer. Cumulatively, 84 fetal cells were recorded by analysis of 5640 cells. The hematopoietic lineages involved in the production of the fetal cells in culture were not assessed. For the cultures established with the 1119 Ficoll layer, the involvement of progenitors or precursors of the erythroid lineage was assumed because postculture sorting was directed toward cells expressing the erythropoietin receptor. <b>Conclusion:</b> We conclude that culturing total nucleated cells from maternal blood is a new procedure that could prove valuable in the detection of the main fetal aneuploidies affecting pregnant populations. (Am J Obstet Gynecol 2000;183:222-5.)

Fetal cells in maternal blood: the use of primed in situ (PRINS) labelling technique for fetal cell detection and sex assessment.

Prenatal diagnosis is presently performed following invasive procedures with variable risks of fetal loss; non-invasive procedures using fetal cells in maternal blood would be welcome for the early detection of fetal sex or aneuploidy. We describe a simple and rapid protocol to detect fetal cells and thus to assess fetal sex. In a first step, nucleated blood cells were separated into mononuclear and polynuclear cells using a double density gradient centrifugation. In a second step primed in situ (PRINS) labelling technique was performed to label Y-chromosomes. 15 samples were studied and correct gender assignment was made in 13/15. The number of labelled nuclei was higher in polynuclear cell phases than in mononuclear cell phases. Moreover, the polylobular aspect of labelled nuclei from polynuclear cell phases strongly suggested that they could belong to fetal polynuclear cells. The PRINS technique combines some advantages of FISH, such as visual assessment of in situ chromosome labelling and the powerful specificity and sensitivity of PCR. In association with a simple enrichment procedure it constitutes a rapid protocol for fetal cell detection, non-invasive early prenatal sex assessment, and could further be applied to detect the main viable aneuploidies.