Use of two different deoxyribonucleic acid probes to detect Y chromosome deoxyribonucleic acid in subjects with normal and altered Y chromosomes

Four experiments evaluated the sensitivity and specificity of molecular techniques to detect human Y chromosome deoxyribonucleic acid. In experiment I, electrophoretic separation of normal male deoxyribonucleic acid fragments after digestion with endonuclease Hae III revealed two male-specific bands of 3.4 and 2.1 kilobase (kb). These bands were not visible if the fraction of male deoxyribonucleic acid in mixed samples was less than 0.3. In experiment II, by means of a repetitive copy Y deoxyribonucleic acid probe (pS4) mapped to Yq12, a male-specific 2.3 kb band was detectable in mixtures of 2.5 ng of male deoxyribonucleic acid and 997.5 ng of 45,X female deoxyribonucleic acid. In experiment III, hybridization with the pS4 probe was performed on the deoxyribonucleic acid of 20 subjects with a normal or a variant Y chromosome. In experiment IV, deoxyribonucleic acid from the same subjects was hybridized to a single copy probe (4B-2) mapped to the Yq11 region. Deoxyribonucleic acid from category A subjects (n = 8) with cytologically normal Y chromosomes hybridized to both deoxyribonucleic acid probes. Deoxyribonucleic acid from category B subjects (n = 2), including a variant Y chromosome that was negative for Q-banding but positive for C-bands, hybridized with the distal pS4 and proximal 4B-2 probes. Deoxyribonucleic acid from category C subjects (n = 10) with variant Y chromosomes uniformly negative for Q- and C-bands, did not hybridize with the pS4 probe. Deoxyribonucleic acid from three of the 10 category C subjects did hybridize to the more proximal sequence-detecting 4B-2 probe. Deoxyribonucleic acid from the remaining seven subjects in category C did not hybridize with either of the deoxyribonucleic acid probes.     

Trophoblast from anembryonic pregnancy has both a maternal and a paternal contribution to its genome.

In a series of 14 anembryonic pregnancies, deoxyribonucleic acid from trophoblast was examined with locus-specific minisatellite probes. It was found in each case that trophoblast from anembryonic pregnancy has both a maternal and a paternal contribution to its genome. This means that although anembryonic pregnancy shares characteristics with complete hydatidiform mole and androgenetic development in the mouse in that there is no embryo, it does not arise by the same genetic process. Of three anembryonic pregnancies that were successfully karyotyped, two had normal female 46,XX chromosome constitutions and one had an abnormal 47,XX + 16 complement. The sex of the trophoblast in each anembryonic pregnancy was determined with a deoxyribonucleic acid probe with Y-specific bands. A male-to-female ratio of 6:8 was found, which is not significantly different from normal.     

Use of human alpha-satellite deoxyribonucleic acid to detect Y-specific centromeric sequences

The Y alphoid deoxyribonucleic acid probe Y97 has proved to be specific for the human Y centromere and to define a Y-specific 5.5 kb Eco RI fragment. Three experiments were designed to evaluate the sensitivity and the specificity of this Y alphoid probe Y97. In the first experiment the centromeric Y-specific 5.5 kb Eco RI fragment was clearly seen in the mixture of 0.050 microgram of male DNA with 4.950 micrograms of female DNA (1%). In the second experiment the same dilutional study was applied to the Yq11-related probe 4B-2 for comparison purpose. In the third experiment, hybridization with the Y97 probe was performed on 20 subjects with mosaic cell lines containing a cytogenetically identifiable Y (n = 10) and a cytogenetically unidentifiable minute (n = 10) fragment. Nineteen of the 20 subjects demonstrated the Y-specific 5.5 kb Eco RI hybridization band with the centromeric Y97 probe. These experiments demonstrated the utility of the Y97 probe to consistently identify cytogenetically altered Y chromosome fragments and confirm the mapping of the alphoid repeat sequences to the centromeric region of the Y chromosome.     

Y-specific sequences in Turner syndrome]

Turner Syndrome (TS) is the only one monosomy that occurrs+ in humans. The cytogenetics of TS is very well known from years. It has been estimated that almost 98-99% of TS foetuses end in abortion. It was suggested that the monosomy arises relatively late during embryonal development and survived TS individuals could be mosaics. It has been proved that mosaic karyotype mos 45,X/46X, + mar(Y) occurrs++ in 2% to 11% of TS patients. The patients having additional cell line containing der(Y) are at increased risk of gonadoblastoma development. In these cases gonadectomy should be considered. Therefore detection of mosaic and establishing the origin of marker chromosome (specially containing Y-specific sequences) is of special importance. The aim of present study was to detect the small mosaics, containing mar(Y) in TS patients, by using PCR and FISH techniques. Eight Y sequences for the PCR analyses as well as bicolor in situ hybridisation with painting probes for Y and X chromosomes have been applied. The positive amplification for Y-specific sequences has been detected in 7% of TS patients. Our results support the thesis that searching for the Y sequences should be introduced to routine genetic TS diagnosis.     

Rapid determination of fetal sex by deoxyribonucleic acid amplification of Y chromosome-specific sequences

Rapid fetal sex tests use either dot blot hybridization to Y chromosome-specific (male) repeat sequences or polymerase chain reaction deoxyribonucleic acid amplification of these Y-specific sequences. We have performed 35 fetal sex determinations, 16 by dot blot alone, 13 by polymerase chain reaction alone, and 6 by dot blot and polymerase chain reaction on samples of fetal blood, amniotic fluid, and chorionic villi. All results have been confirmed by karyotyping. Dot blots have given false-positive "male" results three times. In contrast, the polymerase chain reaction has correctly determined fetal sex in every case, even when the dot blot was in error. The Y-specific polymerase chain reaction has been applied to fetal deoxyribonucleic DNA with a chromosome 15; Y translocation to identify the origin of the translocated material. Thus the polymerase chain reaction appears to be a reliable method to rapidly determine fetal sex that also can be used diagnostically to identify translocated Y-chromosomal material.     

HLA-G expression in trophoblast cells circulating in maternal peripheral blood during early pregnancy

OBJECTIVE: The aim of this study was to assess the use of circulating trophoblast cells in maternal peripheral blood for noninvasive prenatal diagnosis of numeric chromosomal aberrations. STUDY DESIGN: A combined procedure for immunocytochemical identification and deoxyribonucleic acid fluorescence in situ hybridization was used after a single enrichment step consisting of density gradient centrifugation. A specific HLA-G monoclonal antibody was used in combination with X and Y chromosome specific probes in deoxyribonucleic acid fluorescence in situ hybridization to confirm fetal identity of cells bearing HLA-G in the case of a male fetus. RESULTS: We detected fetal trophoblast cells expressing HLA-G in maternal blood starting at 9 weeks' gestation. In addition to fetal sex prediction with X and Y chromosome-specific probes, fetal aneuploidy was confirmed in peripheral blood from a pregnancy complicated by trisomy 21. CONCLUSION: Although the numbers of fetal cells were extremely low, the proof of concept was demonstrated. Early noninvasive prenatal screening for numeric chromosomal abnormalities with fetal trophoblast cells is feasible.     

Molecular identification of chromosome Y sequences in Brazilian patients with Turner syndrome

The investigation of Y-specific sequences in patients with Turner Syndrome (TS) with karyotype 45,X or mosaic, has a fundamental role in the clinical management of these patients. The relationship between the presence of Y chromosome fragments and a higher risk of gonadoblastoma in TS has already been established. The aim of the study was to investigate the presence of Y-chromosome fragments in a population of 42 female Brazilian patients with TS from Mato Grosso state. Cytogenetic analysis has shown the karyotypes 45,X in 27 of them (64.3%) and mosaic in 15 (35.7%). The presence of the Y-primers SRY, DYZ3, ZFY, DYZ1, DYS1 and PABY was investigated in all patients. These markers were amplified by polymerase chain reaction (PCR) technique, using DNA genomic from peripheral blood lymphocytes. None of these patients had shown any Y-chromosome fragments when they were analysed only by the classic cytogenetic technique. The PCR analysis with the Y-specific sequences ZFY and DYZ3 were identified in two different patients (4.8%), both with karyotype 45,X. It was concluded that PCR is efficient in the investigation of hidden Y-fragments in TS patients. Therefore, this method should be included in the routine assistance of these patients.     

Deoxyribonucleic acid hybridization analysis for the detection of urogenital Chlamydia trachomatis infections in women

The presence of Chlamydia trachomatis-related deoxyribonucleic acid sequences in endocervical specimens of 317 women was analyzed by deoxyribonucleic acid hybridization techniques with deoxyribonucleic acid from C. trachomatis used as probes. Samples from 56 of 172 high-risk patients (32.6%) and 16 of 145 low-risk patients (11.0%) contained C. trachomatis-related deoxyribonucleic acid sequences. Direct detection of chlamydial antigen with enzyme-linked immunoassay on the same patients yielded positive rates of 26.3% and 7.3% for the high- and low-risk patients, respectively. C. trachomatis culture confirmed 86.3% of deoxyribonucleic acid-positive results and 84.0% of antigen-positive results. The overall sensitivities of chlamydial deoxyribonucleic acid and antigen assays were 91.7% and 68.8%, respectively, whereas the specificities were 95.3% and 94.7%. Results also suggested that the test of the C. trachomatis deoxyribonucleic acid correlated better with the female urogenital chlamydial infections than did the antigen test of C. trachomatis. The combined results of higher sensitivity in detecting the microorganism and better correlation with disease activity may make the deoxyribonucleic acid hybridization test a useful tool for the early and accurate diagnosis of C. trachomatis infections in female patients.     

Deoxyribonucleic acid analysis of the zinc finger Y gene in 45,X/46,XY subjects

The deoxyribonucleic acid from nine subjects with a 45,X/46,XY karyotype with a cytogenetically intact Y chromosome and phenotypically presenting with bilateral streak gonads, streak and testis, or bilateral scrotal testes along with a control male and female were analyzed for the presence of the zinc finger Y sequence through the molecular probe pDP1007. This particular probe is thought to constitute part of the putative testicular-determining factor gene. All the study subjects demonstrated the presence of zinc finger Y. Laser densitometry studies confirmed a correlation between the intensity of the zinc finger Y band and the percentage of Y cell lines. This study supports the fact that individuals with mixed gonadal dysgenesis and cytogenetically intact Y chromosomes will tend to have intact zinc finger Y sequences.     

Influence of gestational age on fetal deoxyribonucleic acid retrieval in maternal peripheral blood

OBJECTIVE: We wanted to verify whether gestational age influences the retrieval of fetal deoxyribonucleic acid in maternal blood to identify the best period for maternal blood sampling for a future noninvasive prenatal diagnoses. STUDY DESIGN: We amplified 81 deoxyribonucleic acid samples extracted from the peripheral blood of 27 pregnant women (18 bearing male fetuses and 9 bearing females) by nested polymerase chain reaction of the Y-specific sequence DYS14. We obtained three blood samples (one per gestational trimester) from each woman. Statistical evaluation was assessed by the McNemar test of symmetry. RESULTS: Polymerase chain reaction results in male-bearing pregnancies differed significantly between the first and second trimesters and between the second and third trimesters (p < 0.025) in parallel with a decrease in sensitivity in the second trimester (67%) compared with the first (94%) and third trimesters (100%). CONCLUSIONS: The drop in sensitivity from the first to the second trimester witnesses a variable concentration of fetal cells in maternal blood, with a negative balance in the second trimester. Therefore, to achieve an adequate polymerase chain reaction accuracy, the choice of gestational age is relevant and the first trimester seems to be more suitable than the second trimester.(Am J Obstet Gynecol 1997;177:22)     

Mechanism for human papillomavirus transmission at birth

We attempted to investigate mechanisms, in addition to sexual contact, by which human papillomaviruses associated with anogenital tract lesions could be transmitted. Samples of exfoliated cervical cells were obtained from 45 pregnant women and were assayed by Southern blot hybridization analysis for the presence of human papillomavirus nucleic acids. Twenty-five of the 45 women had cells positive for human papillomavirus deoxyribonucleic acid. A neonatal nasopharyngeal aspirate was obtained at term and analyzed for the presence of human papillomavirus deoxyribonucleic acid. We documented the presence of human papillomavirus deoxyribonucleic acid in the oral pharyngeal cavity of the neonates in 15 of 45 nasopharyngeal samples analyzed. Amniotic fluid was obtained from 13 patients when their membranes were artificially ruptured. These samples were assayed for the presence of human papillomavirus deoxyribonucleic acid; two of the 13 amniotic fluid samples contained human papillomavirus deoxyribonucleic acid. The detection of human papillomavirus deoxyribonucleic acid in the oral cavity of neonates is indicative of a perinatal mechanism of viral transmission. The detection of human papillomavirus deoxyribonucleic acid in the amniotic fluid may suggest an in utero mechanism of transmission. However, problems encountered in collecting the amniotic fluid samples preclude us from definitive interpretation of these data.     

Prevalence of low and high risk human papillomavirus types in cervical cells from Hong Kong pregnant Chinese using filter in situ hybridization

Summary Filter in situ hybridization, using separate probes for human papillomavirus deoxyribonucleic acid types 6, 11, 16 and 18, was used to determine the prevailing HPV types amongst a group of pregnant Chinese women. This group had been previously identified using a mixture of probes for HPV types 6, 11, 16, 18, 31, 33, 35 (Virapap^™). Specimens from six of the eleven cases contained one HPV type (55%) while five were positive for two types (45%). The “low-risk” types (HBV6, 11) were identified as frequently as the “high-risk” types (HPV16, 18) in this study.     

Prenatal identification of a Y-chromosome deletion by Y-specific single copy DNA probes

A sex chromosome deletion was identified in the course of prenatal diagnosis for maternal age. Ultrasound pictures revealed male fetal sex and a comparison with the father's Y chromosome suggested that the altered chromosome might be a de novo deletion of the Y chromosome. DNA hybridization with five human Y-specific probes shows that, among the Y-specific sequences recognized by the probes, only two of them are absent. The normal infant, at birth, was mosaic 46, XYq-/46,XY.     

The association between human papillomavirus deoxyribonucleic acid status and the results of cytologic rescreening tests in young, sexually active women.

We examined the utility of cytologic rescreening tests in women who had positive test results for human papillomavirus deoxyribonucleic acid but who were diagnosed as having benign conditions at cytologic testing. One hundred twenty-five Papanicolaou smears from women who were screened for human papillomavirus deoxyribonucleic acid were sent routinely to a private laboratory for diagnoses. These slides were then reviewed independently by two pathologists who were blinded to the human papillomavirus deoxyribonucleic acid results. The effects of cytologic rescreening in cases of both positive and negative human papillomavirus deoxyribonucleic acid were assessed by calculating z scores. Cervical intraepithelial neoplasia was diagnosed in 40% by pathologist A and in 20% by pathologist B of the human papillomavirus-positive subjects compared with none diagnosed by the private cytology laboratory (z = 3.09, p less than 0.005 and z = 1.98, p less than 0.05, respectively). No significant differences were found in the human papillomavirus-negative group. We conclude that cytologic rescreening in human papillomavirus deoxyribonucleic acid-positive women who were initially diagnosed as having benign cytologic results will yield a significant proportion of cases of cervical intraepithelial neoplasia.     

Human papillomavirus infection of the cervix detected by cervicovaginal lavage and molecular hybridization: correlation with biopsy results and Papanicolaou smear

Human papillomaviruses have previously been identified by molecular hybridization in the majority of dysplastic and cancerous lesions of the cervix. Since human papillomavirus types 16 and 18 have been strongly associated with cervical cancer, the identification of patients infected with these specific human papillomavirus types may provide useful prognostic information. We have developed a painless, noninvasive cervicovaginal lavage technique to collect exfoliated cervicovaginal cells, which can be reliably analyzed for the presence of human papillomavirus deoxyribonucleic acid by Southern blot analysis with the use of deoxyribonucleic acid cloned from human papillomaviruses 6, 11, 16, and 18. In a prospective study of 60 women referred to a colposcopy clinic for evaluation of abnormal Papanicolaou smears, we have detected human papillomavirus deoxyribonucleic acid in 16 of 17 (94%) women with a Class III (dysplasia) or IV (carcinoma in situ) Papanicolaou smear, five of 11 (45%) women with a Class II (atypical) Papanicolaou smear, and 10 of 34 (29%) women with a normal Papanicolaou smear. Detection of human papillomavirus deoxyribonucleic acid in cervicovaginal cells was indicative of a dysplastic cervical lesion in 19 of 20 (95%) patients irrespective of Papanicolaou smear results. We conclude that human papillomavirus deoxyribonucleic acid analysis in cervicovaginal cells is a sensitive method to detect dysplastic lesions of the cervix and may be useful in identifying patients with specific types of human papillomavirus infection, who are at risk to develop cervical cancer.     

Detection of cervical Chlamydia trachomatis and Neisseria gonorrhoeae with deoxyribonucleic acid probe assays in obstetric patients.

OBJECTIVE: The Gen-Probe PACE 2 deoxyribonucleic acid probe assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae are targeted against the ribosomal ribonucleic acid of each pathogen. Our study compared the performance of the probe assays with culture for Chlamydia trachomatis (246 patients) and Neisseria gonorrhoeae (310 patients) while screening obstetric patients. STUDY DESIGN: Using culture as a gold standard, we assessed the sensitivity, specificity, positive predictive value, and negative predictive value of the chlamydia and gonorrhea probes. RESULTS: The prevalence of chlamydia by culture was 13.4% and gonorrhea 4.8%. Against culture, the chlamydia probe assay performed as follows: sensitivity 93.9%, specificity 99.1%, positive predictive value 93.9%, and negative predictive value 99.1%. Values for the gonorrhea probe assay were 93.3%, 99.0%, 82.4%, and 99.7%, respectively. Additional molecular analysis of probe-positive-culture-negative specimens suggests that the gonorrhea probe-positive predictive value may be even higher. CONCLUSION: The Gen-Probe PACE 2 deoxyribonucleic acid probe assays for chlamydia and gonorrhea appear to be promising as convenient, reliable, and cost-effective alternatives to conventional cultures in screening obstetric patients.     

Human papillomavirus deoxyribonucleic acid in cervical carcinoma from primary and metastatic sites

Tissue from 13 cervical cancers and pelvic or para-aortic lymph nodes from the same patient were evaluated by deoxyribonucleic acid hybridization with a human papillomavirus type 16 deoxyribonucleic acid probe for the presence of human papillomavirus-related deoxyribonucleic acid sequences. Twelve of the primary malignancies were squamous cancers and one was an adenocarcinoma. Eight of the primary tumors contained human papillomavirus type 16 deoxyribonucleic acid sequences, and five contained viral sequences closely related to human papillomavirus type 16. Histopathologic diagnosis confirmed malignant cells in six of 13 lymph nodes; three of these specimens contained human papillomavirus type 16 sequences while three had human papillomavirus type 16-related sequences. One lymph node that failed to show malignant cells also contained human papillomavirus type 16 deoxyribonucleic acid. The remaining lymph nodes did not contain malignant cells by either histologic examination or deoxyribonucleic acid hybridization. The human papillomavirus deoxyribonucleic acid sequences in the lymph nodes were similar to those in the matched primary cancer in all cases. These data provide further evidence implicating human papillomavirus in the etiology of cervical cancer.     

Evaluating sex chromosome content of sorted human sperm samples with use of dual-color fluorescence in situ hybridization

OBJECTIVE: Although most methods for selecting the sex of offspring by sorting spermatozoa are ineffective at shifting the ratio of Y- to X-containing cells, some commercial sources continue to offer such services. Our objective was to evaluate commercially “sorted” samples with use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populations, donors, observers, and fluorescence in situ hybridization probes.STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were processed as for insemination. Sperm cells from each total sample or motile subfraction were prepared for fluorescence in situ hybridization by incubation with disulfide-reducing agents to expand sperm nuclei. Two sets of X and Y chromosome–specific, fluorophore-labeled deoxyribonucleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridization efficiency, aneuploidy, and sex chromosome content were evaluated in subsets of five unsorted, five female-oriented, and five male-oriented samples. Total and motile subfractions were compared with eight samples. Fluorescence in situ hybridization probes were compared in five paired unsorted samples.RESULTS: No differences were detected between washed samples and paired motile subfractions. No differences in hybridization and aneuploidy were detected between groups of sorted samples. The Y/X ratio was significantly different between the sorted groups. However, male-oriented samples had a lower Y/X ratio than female-oriented samples did. Observer and probe choice accounted for small but significant variations that did not alter conclusions about the Y/X ratio for sorted samples.CONCLUSION: In a series of 10 sorted samples from one commercial source, dual-color fluorescence in situ hybridization demonstrated a small but significant shift in the sex chromosome ratios among samples. However, this shift was opposite to that expected by the orientation of the sorted samples. (Am J Obstet Gynecol 1997;176:1172-80.)     

Fetal granulocytes in maternal venous blood detected by in situ hybridization.

Fetal male cells from maternal venous blood were detected by a non-radioactive in situ hybridization method using the biotinylated Y-specific DNA probe pY431. The hybridizations were performed on Ficoll-Paque-isolated nucleated blood cells obtained from 11 pregnant women in the seventh to 31st week of gestation. A Y-specific signal was detected in both granulocytes and lymphocyte-like cells in seven of the 11 women studied. These women gave birth to boys. In one of the four remaining cases, a Y-specific signal was detected in the lymphocyte-like cells but not in the granulocytes. This woman gave birth to a girl. The other three women had no cells with a Y-specific signal and all three gave birth to girls. Altogether, 83,500 nucleated cells were analysed. One hundred and three cells showed a Y-specific signal. Of these Y-specific cells, 62 per cent were granulocytes and 38 per cent lymphocyte-like cells. Our results suggest that fetomaternal transfer of granulocytes is common and that it occurs as early as in the seventh week of gestation. None of the ten non-pregnant female control samples showed positive cells with the Y-chromosome-specific probe; approximately 97 per cent of the cells from the five adult male controls showed a Y-specific signal. Our results indicate that in situ hybridization using a Y-specific DNA probe performed on granulocytes in maternal blood can be used for fetal male sex determination.     

Increased frequency of detection of human papillomavirus deoxyribonucleic acid in exfoliated cervical cells during pregnancy

Exfoliated cells of the uterine cervix obtained from women during pregnancy and at the time of their first postpartum examination were used to monitor the prevalence of human papillomavirus infections in this population and to study the natural fluctuations in viral expression. When deoxyribonucleic acid hybridization analysis alone was used to monitor the presence of human papillomavirus infection, 20.9% of our study population had results that were positive for human papillomavirus deoxyribonucleic acid during their first-trimester examinations. A dramatic increase in the percentage of women with positive results for human papillomavirus deoxyribonucleic acid was observed at the time of the patients' third-trimester examinations (46%). The overall increase in human papillomavirus-positive patients was a combination of a small number of patients who had positive results on their first examination and negative results on their second examination, and a larger number of patients who had negative results on their first-trimester examination and positive results for human papillomavirus deoxyribonucleic acid in the exfoliated cervical cells at the time of their third-trimester examination. The total percentage of patients with positive results for human papillomavirus deoxyribonucleic acid in their cervical cells at one or both assay points during pregnancy was 52.5%. Samples obtained at the postpartum examination demonstrated a dramatic decrease in the number of samples positive for human papillomavirus deoxyribonucleic acid (17.5%). This result was a combination of a large decrease in human papillomavirus-positive patients coupled with a small increase in detectable levels of human papillomavirus deoxyribonucleic acid in cervical samples from patients who had negative results on their previous examination. This study demonstrates a very high level of detectable human papillomavirus deoxyribonucleic acid in exfoliated cervical cells obtained during pregnancy and shows that the detectable levels of human papillomavirus deoxyribonucleic acid fluctuate during pregnancy.