Dental pulp fibroblast and sex determination in controlled burial conditions / Fibroblastos de la pulpa dental y la determinación del sexo en condiciones controladas de enterramiento

The need to identify bodies that are found as a result of disappearances with a diversity of causes, illegal burials and massive disasters, represent a wide percentage of dentistry practice on forensic research. The following study determined the performance of Barr Body Test, in fibroblasts of healthy teeth, under different conditions of burial (in vitro) with variations in pH, humidity and salinity in terms of general accuracy and sensitivity for men and women. Analyzed sample considered 47 dental pulps, taken from teeth under burial conditions during a period of a month. From dental pulps samples, 265 histological cuts valid for this study, were obtained, which were observed with an optical microscope under conventional H/E staining. Results showed a 98.9% of well-diagnosed cases, which correspond to the overall accuracy of the method. Sensitivity for men was 97.5% and 100% for women, over the analyzed sample. In low humidity conditions, 3 samples of badly diagnosed cases in men were observed, with a group accuracy of a 90%, with a sensitivity of 25% for men and 100% for women. The present study establishes that based on these results, the performance of Barr Body Test in fibroblasts, proposed for healthy pulp teeth, is not affected by burial conditions in terms of pH (acid-alkaline), salinity (high-low) and high humidity.

La necesidad de identificar cuerpos que resultan como consecuencia de desapariciones de causas variadas, inhumaciones ilegales y desastres masivos representa un porcentaje amplio en el quehacer odontológico en un escenario de investigación forense. El presente estudio determinó el rendimiento de la prueba diagnóstica de observación del cuerpo de Barr en células de la pulpa de dientes sanos, sometidos a distintas condiciones de enterramiento (in vitro) con variación de pH, humedad y salinidad en términos de exactitud general y sensibilidad para hombres y mujeres. La muestra analizada consideró 47 pulpas dentales, extraídas de dientes sometidos a condiciones de enterramiento durante un mes. De las pulpas dentarias se obtuvieron 265 cortes histológicos válidos para el estudio, los cuales mediante la tinción convencional H/E, fueron observados al microscopio óptico. Los resultados arrojaron un 98,9% de casos bien diagnosticados, que correspondió a la exactitud general del método. La sensibilidad para hombres fue de 97,5% y para mujeres de 100% sobre el total de la muestra analizada. Las condiciones de pH (ácido y alcalino), salinidad (alta y baja) y alta humedad presentaron una exactitud de grupo de 100%, con una sensibilidad para hombres y mujeres de 100%. En la condición de baja humedad se observaron 3 muestras de hombres mal diagnosticadas con una exactitud de grupo de 90% y sensibilidad para hombres de 25% y para mujeres de 100%. A la luz de los resultados, el presente estudio establece que el rendimiento de la prueba diagnóstica de observación del cuerpo de Barr en fibroblastos, propuesto para pulpas de dientes sanos, no se afecta con las condiciones de enterramiento propuestas bajo pH ácido ­ alcalino, salinidad alta ­ baja y humedad alta.

Reliability of sexual dimorphism in blood.

BACKGROUND: Demonstration of sex chromatin forms an important aspect of human genetics. It also establishes the interrelationship between sex chromatin and an inactive X-chromosome. The term "sex chromatin" in blood refers to the "Drumsticks of polymorphonuclear leukocytes" or "Davidson's bodies". OBJECTIVE: This correlative study evaluates the presence of these drumsticks quantitatively and also highlights the concept of blood chimaerism in humans. METHOD: Leishman-stained peripheral blood smears from 60 individuals (30 males and 30 females) were obtained and studied under bright-field microscope (40X) for presence of Drumstick appendages. RESULTS AND CONCLUSION: On comparing mean numbers of Davidson's bodies in females and males, an extremely significant correlation (P < 0.0001) was seen. Hence, it could be surmised that the presence of appendages in neutrophils (Drumstick bodies) can be useful in gender differentiation.

Observation of sex chromatin in exhumed bones, evaluation of their diagnosis value / Observación de la cromatina sexual en osamentas exhumadas, evaluación de su valor diagnóstico

Observation of sexual chromatin has shown to be very helpful in gender forensic diagnosis. In the present study we analyzed the diagnosis performance of the method in, non-treated or treated with conventional bone techniques, exhumed bone pieces. We used long bones of male and female individuals, the method applied is described in Suazo et al. (2010). In the non-treated exhumed pieces, the general accuracy of the method was 75 percent, while in the treated pieces the method was inapplicable due to the lack of cells in the tissue. Our results suggest that it is possible to determine the sex of aged human bones buried under different conditions through a fast and simple histological method, but the treatment with physical and chemical means eliminates the remaining cells in the bone tissue.

La observación de la cromatina sexual ha demostrado ser útil en el diagnóstico forense del sexo. En este estudio analizamos el rendimiento diagnóstico del método en piezas óseas exhumadas no tratadas y tratadas mediante osteotécnica convencional. Utilizamos muestras de huesos largos de individuos de sexo masculino y femenino, el método se aplicó de acuerdo a lo descrito por Suazo et al., (2010). En las piezas exhumadas no tratadas la exactitud general del método fue del 75 por ciento, mientras que en las piezas tratadas el método resultó inaplicable, debido a la ausencia de células en el tejido. Nuestros resultados sugieren que es posible determinar el sexo en osamentas humanas exhumadas de larga data y en diferentes condiciones de enterramiento, mediante un método histológico rápido y sencillo, pero que el tratamiento por medios físicos y químicos elimina las células remanentes en el tejido óseo.

A top-down analysis of Xa- and Xi-territories reveals differences of higher order structure at ≥ 20 Mb genomic length scales.

The active and inactive X (Xa;Xi) territory with its seemingly highly compacted Barr body in nuclei of female mammalian cells provide a key example for studies of structure/function relationships in homologous chromosomes with different functional properties. Here we used about 300 human X-specific large insert clones to generate probe sets, which target physically or functionally defined sub-chromosomal segments. We combined 3D multicolor FISH with quantitative 3D image analysis in order to compare the higher order organization in Xi-and Xa-territories in human diploid fibroblasts (HDFs) at various length scales ranging from about 50 Mb down to 1 Mb. Xi-territories were characterized by a rounder shape as compared to the flatter and more extended shape of Xa-territories. The overall compaction of the entire Xi-territory, including the Barr body, was only 1.2-fold higher than the Xa-territory. Significant differences, however, were noted between distinct subchromosomal segments: At 20 Mb length scales higher compaction in Xi-territories was restricted to specific segments, but higher compaction in these segments was not correlated with gene density, transcriptional activity, LINE content or histone markers locally enriched in Xi-territories. Notably, higher compaction in Xi-territories observed for 20 Mb segments was not reflected accordingly by inclosed segments of 1-4 Mb. We conclude that compaction differences result mainly from a regrouping of ~1 Mb chromatin domains rather than from an increased condensation of individual domains. In contrast to a previous report, genes subject to inactivation as well as escaping from inactivation were not excluded from the interior of the Barr body.

Evaluación funcional del esperma en varones fértiles columbianos / Functional evaluation of sperm in colombian fertile men

OBJETIVO: El fin del presente estudio fue evaluar en 111 eyaculados de individuos fértiles la integridad de la membrana de los espermatozoides antes de la selección, y la movilidad, la concentración, la integridad de la cromatina antes y después de la selección espermática. MÉTODOS: Evaluamos la integridad de la membrana (usando la prueba de hinchazón hipoosmotica y eosina Y) antes de la selección, y la integridad de la cromatina (usando naranja de acridina) concentración y movilidad antes y después de la separación mediante la técnica de migración sedimentación. RESULTADOS: Los datos de la integridad de la membrana espermática mediante la prueba de hinchazón hipoosmótica y eosina Y no mostraron diferencias estadísticamente significativas y la correlación entre estas fue baja. El porcentaje de espermatozoides móviles (grado a + b) se incrementó de 57% a 87% (p < 0.001), la concentración disminuyó de 89 a 31 x 106 espermatozoides/ mL (p < 0.001) y la integridad de la cromatina se incrementó significativamente (p < 0.0001) antes y después de la separación. CONLCUSIONES: la gran variación en los valores obtenidos en las pruebas funcionales en hombres fértiles requiere una re-evaluación del uso de estas pruebas en la práctica clínica de infertilidad

OBJECTIVE: The aim of the present study was to evaluate in 111 ejaculates from fertile men membrane integrity of spermatozoa before selection and sperm motility, and sperm concentration and chromatin integrity before and after selection of motile spermatozoa. METHODS: We evaluated the membrane integrity (using hypoosmotic swelling test and Eosin-Y) before separation and chromatin integrity (using acridine orange), concentration and motility before and after separation by migration sedimentation technique. All individuals had pregnant wives or had procreated a baby during the last year. RESULTS: The data of sperm membrane integrity by the eosin-Y and hypoosmotic swelling tests did not show significant statistical differences and the correlation between them was low. The percentage of motile sperm (grades a + b) increased from 57% to 87% (p < 0.001), the concentration decreased from 89 to 31 x 106 sperm/mL (p < 0.001) and chromatin integrity increased significantly (p < 0.0001) after separation of semen. CONCLUSIONS: The great variation in the values obtained in the functional test in fertile males requires a re-evaluation of the use of these tests in clinical practice of infertility

Value of DNA integrity assays for fertility evaluation.

DNA damage in the male germ line is associated with failed fertilisation, impaired preimplantation development and poor pregnancy outcomes, whether the insemination is natural or artificial. It is also apparent that DNA damage in spermatozoa is associated with adverse impacts on the health and wellbeing of children. This may be particularly important in the case of conceptions involving intracytoplasmic sperm injection. With this technique DNA damaged spermatozoa that would, under physiological circumstances, be excluded from the conception process are able to initiate pregnancies. Although the oocyte actively repairs the DNA damage brought into the zygote by the fertilising spermatozoon, errors in this repair process would generate mutations that might in turn be linked to the increased incidence of dominant genetic disease and childhood cancer seen in the offspring of fathers possessing DNA damaged spermatozoa. Significantly, the incidence of such mutations is so low that it might be several generations before the full consequences of using DNA damaged spermatozoa in assisted conception cycles are realised. The factors modulating DNA damage in the male germ line are complex and largely unresolved. They involve advanced paternal age, exposure to xenobiotics and male genital tract infection. The types of damage being measured by the assays used in most laboratories (SCSA, Comet and TUNEL) are also uncertain. Molecular characterization of this DNA damage might provide insights into the underlying aetiologies, facilitate the development of optimised diagnostic methods for its detection and suggest logical avenues to pursue for its correction and ultimate prevention.

Analysis of sperm DNA fragmentation using flow cytometry and other techniques.

The goal of a rapid, precise and objective sperm measure of sperm DNA fragmentation, in situ, was realised a quarter century ago by the pioneer development of the sperm chromatin structure assay (SCSA). Since then, measurements on many thousands of sperm samples from a variety of animals and humans exposed to reproductive toxicants and having different diseases or infertility pathologies have solidly established the utility of sperm DNA fragmentation in experimental and clinical settings. New methods have been developed that measure sperm DNA fragmentation by different means such as the enzymatic labelling of broken DNA strands (Tunel assay by light microscopy or flow cytometry) and the light microscope observations of DNA fragments in the Comet assay and the negative loops of sperm chromatin in the Halo sperm assay. These assays have proven useful in the areas of livestock and captive wildlife reproduction efficiency, toxicology and more recently in the human infertility clinic. A significant problem is the lack of quality control and standards of threshold values between laboratories. At this time, only the SCSA has been deemed sufficiently rigorous in methodology and established clinical thresholds for utility in the human infertility clinic.

Probing the W chromosome of the codling moth, Cydia pomonella, with sequences from microdissected sex chromatin.

The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells. We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and start an analysis of the W-chromosome sequence composition. With fluorescence in situ hybridization (FISH), the euchromatin probe labelled all chromosomes, whereas the W-chromatin DNA proved to be a highly specific W-chromosome painting probe. For sequence analysis, DOP-PCR-generated DNA fragments were cloned, sequenced, and tested by Southern hybridization. We recovered single-copy and low-copy W-specific sequences, a sequence that was located only in the W and the Z chromosome, multi-copy sequences that were enriched in the W chromosome but occurred also elsewhere, and ubiquitous multi-copy sequences. Three of the multi-copy sequences were recognized as derived from hitherto unknown retrotransposons. The results show that our approach is feasible and that the W-chromosome composition of C. pomonella is not principally different from that of Bombyx mori or from that of Y chromosomes of several species with an XY sex-determining mechanism. The W chromosome has attracted repetitive sequences during evolution but also contains unique sequences.

Clinical aspects of sperm DNA fragmentation detection and male infertility.

Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. Currently, there are four major tests of sperm DNA fragmentation, including the Comet, Tunel, sperm chromatin structure assay (SCSA) and the acridine orange test (AOT). The Comet assay is a light microscope technique where the sperm cells are mixed with melted agarose and then placed on a glass slide. The cells are lysed and then subjected to horizontal electrophoresis. The Tunel assay, another light microscope technique, transfers labeled nucleotide to the 3'OH group of a broken DNA strand with the use of terminal deoxynucleotidyl transferase. The fluorescence intensity of each scored sperm is determined as a "yes" or "no" for sperm on a light microscope slide or by channels of fluorescent intensity in a flow cytometer. The light microscope-based AOT, uses the metachromatic properties of acridine orange to stain sperm cells. The SCSA treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA as measured by flow cytometry (FCM) as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). The SCSA method has defined a 27-30% DNA fragmentation index (DFI) as the point in which a man is placed into a statistical category of taking a longer time to in vivo pregnancy, intra uterine insemination (IUI) and more routine in vitro fertilization (IVF) cycles or no pregnancy. Current data suggest that intracytoplasmic sperm injection (ICSI) may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.

Clinical and diagnostic features of patients with suspected Klinefelter syndrome.

Klinefelter syndrome, with an incidence of 1:600 male newborns, is the most frequent form of male hypogonadism. However, despite its relatively high frequency, the syndrome is often overlooked. To prevent such oversights, the clinical features should be better characterized, and simple screening tests should be used more frequently. In a cohort of 309 patients suspected of having Klinefelter syndrome, we evaluated the clinical symptoms as well as the diagnostic value of the Barr body test for screening procedures. On the basis of chromosome analysis, 85 patients (group I) were diagnosed as having Klinefelter syndrome, and 224 patients had a 46,XY karyotype (group II). Barr body analysis revealed a specificity of 95% and a sensitivity of 82% for the diagnosis of Klinefelter syndrome. General features (eg, reason for admission, age, age of the parents, body weight, and frequency of maldescended testes) were not different between the groups, except that group I had a higher proportion of patients with a lower educational background. Compared to group II, patients with Klinefelter syndrome were taller (P <.001); had smaller testis volumes (P <.0001), higher follicle-stimulating hormone (FSH) and luteinizing hormone (LH) values; and carried a tendency for less androgenic phenotype and secondary hair distribution. Testosterone, estradiol, sex hormone-binding globulin (SHBG), and prostate-specific antigen (PSA) serum levels as well as prostate volume were not significantly different between the groups. In patients who provided an ejaculate, azoospermia was found in 54% of the patients in group II and in 93% of the patients with Klinefelter syndrome. Although not exclusively characteristic for Klinefelter syndrome, the combination of low testicular volume and azoospermia, together with elevated gonadotropins, is highly indicative for a Klinefelter syndrome and should stimulate further clinical investigations. Barr body analysis provides a quick and reliable screening test, which, however, must be confirmed by karyotyping.

Three-dimensional reconstruction of painted human interphase chromosomes: active and inactive X chromosome territories have similar volumes but differ in shape and surface structure.

This study provides a three-dimensional (3D) analysis of differences between the 3D morphology of active and inactive human X interphase chromosomes (Xa and Xi territories). Chromosome territories were painted in formaldehyde-fixed, three-dimensionally intact human diploid female amniotic fluid cell nuclei (46, XX) with X-specific whole chromosome compositive probes. The colocalization of a 4,6-diamidino-2-phenylindole dihydrochloride-stained Barr body with one of the two painted X territories allowed the unequivocal discrimination of the inactive X from its active counterpart. Light optical serial sections were obtained with a confocal laser scanning microscope. 3D-reconstructed Xa territories revealed a flatter shape and exhibited a larger and more irregular surface when compared to the apparently smoother surface and rounder shape of Xi territories. The relationship between territory surface and volume was quantified by the determination of a dimensionless roundness factor (RF). RF and surface area measurements showed a highly significant difference between Xa and Xi territories (P < 0.001) in contrast to volume differences (P > 0.1). For comparison with an autosome of similar DNA content, chromosome 7 territories were additionally painted. The 3D morphology of the chromosome 7 territories was similar to the Xa territory but differed strongly from the Xi territory with respect to RF and surface area (P < 0.001).

Meiosis in holocentric chromosomes: kinetic activity is randomly restricted to the chromatid ends of sex univalents in Graphosoma italicum (Heteroptera).

We have determined the number and location of the nucleolar organizing regions in spermatocytes of Graphosoma italicum (2n = 12A + XYmale/XXfemale) by means of silver impregnation, chromomycin A3/distamycin A staining and fluorescence in situ hybridization. The identification of only one nucleolar organizing region located at one of the X chromosome ends has provided a suitable cytological marker to analyse the segregation of this univalent and that of the XY pseudobivalent during the first and second meiotic divisions respectively. Our results clearly show that at first meiotic metaphase the chromatids of the X chromosome are orientated with their long axes perpendicular to the polar axis. Although the kinetic activity is restricted to only one end in both X chromatids during the first meiotic division, both ends of the same chromatid have the same probability of showing such kinetic activity. In this sense, we also report that the chromatid segregation may be initiated either at the same sister chromatid ends or at opposite ends in each chromatid. Thus, this indicates a sex chromatid independence as regards to the chromatid segregation during the first meiotic division. Throughout the second meiotic division both ends of the X chromatid are involved with the same probability in the end-to-end association to conform the XY pseudobivalent. This also implies a random localization of the kinetic activity at the ends opposite to those involved in the end-to-end association.

[Activity of W-sex heterochromatin and accumulation of the nuage in nurse cells of the lepidopteran Ephestia]. / Activité de l'hétérochromatine sexuelle-W et accumulation du nuage dans les cellules nourricières du lépidoptère Ephestia.

In the Lepidopteran Ephestia, in the female sex, the W chromosome, with which no function is known to be associated, is, in whole or part, maintained in a heterochromatic state in all cellular categories, except for the oocyte. In the present work, however, W-sex heterochromatin (W-SH) is demonstrated by ultrastructural autoradiography to be transcriptionally active in nurse cells during previtellogenesis. This activity is accompanied by accumulation of "nuage" in the perinuclear cytoplasm, both phenomena arresting at the beginning of vitellogenesis. We have performed kinetic analysis of the labeling associated with W-SH and with nuage through a pulse-chase experiment, together with high resolution examination of the structures visible in the vicinity of active W-SH. Our results suggest that W-SH transcripts are packaged and transported to the cytoplasm within polyparticles typically resembling the hnRNP particles--the structures packaging (pre)messenger RNA which are isolated from the nuclei of numerous Eukaryotes--and that they are concentrated within the nuage upon leaving the nucleus. Such findings explain the close relationship observed in Ephestia, both in space and time, between the nuage and active W-SH. In different organisms, including Drosophila, the nuage has been proposed to be a site of assembly of germ plasm. Considering this hypothesis as well as our results with Ephestia, we speculate that the activity of W-SH detected in nurse cells reflects the expression of one or several genes located within the heterochromatin of the W chromosome, and that the function of these genes is related with the elaboration of germ plasm. The implications raised by these unexpected proposals are mentioned.

Correlation between Barr bodies and neutrophil drumsticks.

There is a marked difference between the frequencies of neutrophil nuclear drumsticks and mucosal cell Barr bodies in any given woman despite the fact that both represent an inactivated X chromosome. We present results of a prospective study carried out on 100 normal Saudi females to assess the statistical correlation between these two variables. We conclude that each is independent of the other. The lack of statistical correlation perhaps relates to maturational and nuclear configuration factors.

Frequency of X-chromatin in pregnant women during the second trimester of gestation

The frequency of X-chromatin was determined in pregnant women during the second trimester of gestation in order to establish possible correlations with the changes in steroid hormone levels occurring during this period and compare the curve with that for normal nonpregnant women. The mean frequency of X-chromatin in oral mucosa cells from a salivary suspension was 19.97% for the 24 patients studied, a statistically higher value than that obtained for nonpregnant women

Frequency of X-chromatin in pregnant women during the second trimester of gestation.

The frequency of X-chromatin was determined in pregnant women during the second trimester of gestation in order to establish possible correlations with the changes in steroid hormone levels occurring during this period and compare the curve with that for normal nonpregnant women. The mean frequency of X-chromatin in oral mucosa cells from a salivary suspension was 19.97% for the 24 patients studied, a statistically higher value than that obtained for nonpregnant women.

[The antenatal diagnosis of the sex of the fetus by the cytological determination of sex chromatin]. / Antenatalna diagnoza pol na ploda chrez tsitologichno opredeliane na poloviia khromatin.

Forty women between 17 and 23 weeks' gestation were included in the study, whose interruption of pregnancy was performed by medical and social indications. The method of Aburel-Atanasov was used as in 10 ml of the removed amniotic fluid sex chromatin was examined cytologically on cellular smears. Staining and determination of sex chromatin was made by the method of Dokumov. The fetus of male sex, when there was no sex chromatin, but the sex was female, when it was present in various percentage of cells. The genuine sex of the fetus was determined after interruption of pregnancy. Coincidence of cytological and genuine sex of the fetus was found in 36 cases (92.31%), but the sex was determined not exactly in 3 cases (7.69%). There was no sufficient material for preparation of high-grade cytological smears in one case. 14 out of 40 fetuses were of female sex (with sex chromatin in 28.57% of cells on the average) and 25 of male sex. The significance and advantages of the method in comparison with other methods for determination of fetal sex at the same term of pregnancy are discussed.

[Movement of corneal endothelium after penetrating keratoplasty. Observation of sex chromatin as a cell marker].

There is a hypothesis that corneal endothelial cells migrate after penetrating keratoplasty from the areas of higher cell density to the lower cell density areas. In order to confirm this hypothesis, the author performed exchange penetrating keratoplasty in normal albino rabbits using four combinations of sex and age and examined the movement of the endothelium between donors and recipients by observing the density and sex chromatin of the endothelial cells. As a result, there was no significant difference of the rate of decrease of the endothelial cell density of the postoperative grafts between young and old grafts. In the experiment using the sex chromatin of the endothelial cells as a cell marker, it was concluded that the endothelium of the rabbit cornea migrate from the young grafts to the old recipients or from the young recipients to the old grafts. In the control study exchanging rabbit grafts between animals of different sex but the same age the count of the sex chromatin of the endothelium of the grafts and recipients did not change postoperatively. The results confirmed the above hypothesis concerning the migration of the transplanted corneal endothelial cells in the rabbit cornea.