Aneuploidy rates for chromosomes X/Y and 18 among preselected spermatozoa in men with severe teratospermia.

Eight infertile men with various degrees of oligoasthenoteratozoospermia and repeated implantation failure were selected for this study due to exceptionally high rates of sperm aneupoidy in their ejaculates. All subjects had normal physical examination, karyotype and serum FSH concentration. Prior to IVF treatment, spermatozoa was collected, processed, micromanipulated and tested for chromosomes X, Y and 18 using fluorescence in-situ hybridization. Aneupoidy rates for chromosomes X, Y and 18 were determined among sperm population selected for normal morphology using high-order magnification light microscopy. A second group of fast motile spermatozoa were collected using an intracytoplasmic sperm injection pipette from the medium-oil interface from microdroplets. The average aneuploidy rates for the three chromosomes were 7.6% (395/5182) in the sperm specimen before selection, 8.7% (116/1326) in the normal morphology selected group and 4.3% (59/1388; P<0.001) in the fast motile selected group. In conclusion, high-magnification light microscopy aimed at selection of spermatozoa with normal morphology did not affect the aneuploidy rate. On the other hand, fast motile spermatozoa harboured significantly less chromosomal abnormalities (P<0.001). Preselection of the most rapid sperm subpopulation for intracytoplasmic sperm injection may improve the qualities of the fertilizing spermatozoon.

Chromosomal integrity of human preimplantation embryos at different days post fertilization.

INTRODUCTION: In order to investigate the dynamics of genomic alterations that occur at different developmental stages in vitro, we examined the chromosome content of human preimplantation embryos by molecular-cytogenetic techniques at the single-cell level, up to 13 days post fertilization. METHODS: The embryos were genetically analyzed several times during their development in culture; each embryo was first analyzed by FISH at 'Day 3' post fertilization, than during its growth in vitro and the third analysis was performed at development arrest, then the entire blastocyst was analyzed by comparative genomic hybridization (CGH/aCGH). RESULTS: We found that while on 'Day 3' only 31% of the embryos were detected as normal, on 'Day 5-6', 44% of the embryos were classified as normal and on 'Day 7', 57% were normal. On 'Days 8-13', 52% of the embryos were classified as chromosomally normal. One third of the embryos that were chromosomally abnormal on 'Day 3', were found to be normal at development arrest point. DISCUSSION: These dynamic changes that occur at early developmental stages suggest that testing a single blastomere at 'Day 3' post fertilization for PGD might inaccurately reflect the embryo ploidy and increase the risk of false aneuploidy diagnosis. Alternatively, blastocyst stage diagnosis may be more appropriate.

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.

Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells.

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

PURPOSE: Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. METHODS: Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. RESULTS: Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. CONCLUSIONS: Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

Fish based preimplantation genetic diagnosis to prevent DiGeorge syndrome.

PURPOSE: To report the performance of fluorescence in-situ hybridization in the setting of preimplantation genetic diagnosis in order to diagnose embryos affected by DiGeorge syndrome. DESIGN: Case report. SETTING: Academic referral center. PATIENT: A 32 year-old female affected by DiGeorge syndrome. INTERVENTION(S): History and physical examination, karyotyping, amniocentesis, preimplantation genetic diagnosis, fluorescence in-situ hybridization. MAIN OUTCOME MEASURE(S): Avoidance of pregnancy with embryo affected by DiGeorge syndrome. RESULT(S): Termination of pregnancy with an affected embryo followed by fluorescence in-situ hybridization based preimplantation genetic diagnosis and delivery of healthy offspring. CONCLUSION(S): The combination of preimplantation genetic diagnosis with fluorescence in-situ hybridization is recommended to prevent pregnancies with DiGeorge syndrome affected embryos in properly selected patients.

Genetic alterations detected by comparative genomic hybridization and recurrence rate in epithelial ovarian carcinoma.

To assess the putative correlation between comparative genomic hybridization (CGH)-detectable genetic alterations in epithelial ovarian cancer and disease recurrence, conventional CGH was performed on 45 epithelial ovarian cancers: 26 tumors from sporadic, BRCA mutation noncarriers and 11 and 8 tumors from BRCA1 and BRCA2 mutation carriers, respectively. Relevant clinical data, including histology, grade, stage, size of residual tumor, recurrence, and survival, were obtained from outpatient and inpatient charts. Among the 45 cases, the most common regions involving gain of DNA copy number were 3q (n = 23; 51%), 8q (n = 21; 47%), and 1q (n = 14; 31%), and the most common regions with loss were 19 and 22 at 9 cases (20%) each, followed by 5q (n = 6; 13%). In multivariate analysis, the total number of genetic alterations was not associated with risk of recurrence, but gain in 5p was associated with a higher risk of recurrence (hazard ratio HR = 6.06, P = 0.0399), and gain in 1p as well as loss in 5q were associated with a significant decrease in recurrence (HR = 0.08, P = 0.0079, and HR = 0.10, P = 0.0143, respectively). Recurrence rate in patients with epithelial ovarian cancer is seemingly associated with specific genetic alterations detected by CGH, but the specific genes involved and the implications of these findings await further studies.

Is sperm donor karyotype analysis necessary?

A case study involving four female patients inseminated with sperm samples from the same donor is reported. Routine amniocentesis of one of the patients revealed that the fetus is a carrier of a balanced reciprocal translocation, 46,XY,t(1;10)(q12;q11.2), which was subsequently detected in the donor. Counselled amniocentesis in the three remaining patients led to the detection of an additional translocation carrier fetus. All four pregnancies resulted in live births. Fluorescence in-situ hybridization was applied to identify normal, balanced or imbalanced sperm cells of the donor. Accordingly, routine karyotype analysis of sperm donors was introduced, and is now recommended in the authors' unit.

Association of the low-activity COMT 158Met allele with ADHD and OCD in subjects with velocardiofacial syndrome.

Velocardiofacial syndrome (VCFS) is caused by a microdeletion in chromosome 22 and is a risk factor for the development of schizophrenia and other psychiatric disorders. The catechol-O-methyltransferase (COMT), residing in the 22q11.2 microdeletion region, is a major candidate gene for genetic susceptibility to neuropsychiatric disorders in VCFS. Individuals with VCFS carrying the low-activity allele (COMTL) are expected to have the lowest possible COMT activity since they have only a single copy of the gene. We explored the possibility that COMTL is associated with psychiatric disorders commonly found in VCFS. Fifty-five unrelated individuals with VCFS underwent psychiatric evaluation and were genotyped for the COMT 158Val/Met polymorphism coding for COMT high/low-activity alleles. The COMTL allele was significantly more prevalent in VCFS subjects with attention deficit hyperactivity disorder (ADHD) (73.9% vs. 33.3%, OR 5.67, chi2=7.76, p=0.005) and obsessive-compulsive disorder (OCD) (78.6% vs. 33.3%, OR 7.33, chi2=7.24, p=0.007) than in the control group (VCFS subjects without OCD, ADHD and schizophrenia/schizoaffective (SZ/SZaff) disorder). The results of this study suggest that greatly reduced COMT activity, as expected in VCFS COMTL individuals may be a risk factor for psychiatric sequelae in this population. Future longitudinal studies focusing on additional COMT polymorphic sites and other candidate genes from the deleted region will elucidate the molecular pathways leading to schizophrenia and other psychiatric disorders in VCFS.

Proximal 19q trisomy: a new syndrome of morbid obesity and mental retardation.

AIMS: To report on the clinical and metabolic characteristic and the unique chromosomal defect of a mentally retarded and morbidly obese patient. METHODS: A 13-year follow-up, including insulin sensitivity, lipid profile and polysomnography studies and various therapeutic interventions are described. The presence of a supernumerary marker in karyotype preparation was further studied by fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) was used to identify the source of the chromosomal marker. RESULTS: Insulin resistance was found by the homeostatic model assessment (HOMA) and the quantitative insulin sensitivity check index (QUICKI). M-FISH identified euchromatin derived from chromosome 19, and CGH confirmed the FISH results and demonstrated that the supernumerary marker derived from 19q12 to 19q13.2. CONCLUSION: The clinical and metabolic characteristics in association with partial chromosomal trisomy differ our patient from the currently known syndromes of obesity and mental retardation. The metabolic impairments in this case can derive from unbalanced expression of several genes in the 19q12-19q13.2 region, genes that are related to adipose tissue homeostasis and insulin resistance. The clinical and genetic similarities to a previously reported case may suggest that partial 19q trisomy is a new syndrome of obesity and mental retardation.

Mapping of a gene causing brittle cornea syndrome in Tunisian jews to 16q24.

PURPOSE: To map the gene that causes brittle cornea syndrome (BCS). METHODS: Five patients from four families, all of Jewish Tunisian origin, were recruited into the study. Four of the five patients had red hair. DNA from the five patients and 104 control chromosomes was typed with seven 16q polymorphic markers surrounding the hair color gene, MC1R. RESULTS: A common haplotype in the homozygous state, comprising five markers spanning 4.7 Mb on chromosome 16q24, was found in all five patients but in none of the control subjects (P < 0.00001). CONCLUSIONS: The gene that causes BCS maps to a 4.7-Mb interval, between the markers D16S3423 and D16S3425 on 16q24.

Genomic analyses of primary and metastatic serous epithelial ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the western world. In 75% of patients, peritoneal metastases are found at the time of primary surgery. However, the genetic events leading to the development of ovarian tumors and to the genetic progression toward metastasis remain unclear. To gain insight into this issue, the types and patterns of DNA copy number changes were compared between primary ovarian tumors and their respective metastases by using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The genetic alterations (deletions and amplifications) detected by CGH were similar in the primary tumors and in their respective metastases. Moreover, the FISH results show a similar pattern of chromosomal abnormalities. Our results imply that the major gross genetic changes in ovarian cancer take place in the primary tumor, and the additional genetic changes that may occur in the metastases are not detectable by CGH.

Analysis of fetal blood cells in the maternal circulation: challenges, ongoing efforts, and potential solutions.

The invasive procedures amniocentesis and chorionic villus sampling (CVS) are routinely applied in pregnancies at risk for fetal abnormalities and the results obtained are the gold standard for prenatal diagnosis. Because these methods of fetal cell procurement involve a 0.5-2% risk for fetal loss, they are recommended mainly in cases at high risk for fetal genetic or cytogenetic abnormalities. The development of a reproducible, reliable, noninvasive method based on retrieval of rare fetal cells from the maternal circulation will render testing feasible for the general population. Despite intensive investigation, a satisfactory, clinically acceptable method has not yet emerged. Several cell types have been targeted to this end, mostly nucleated red blood cells (NRBC), CD34+ hematopoietic progenitors, and trophoblasts. Although these cell types have been unequivocally proven to be present in the maternal circulation, each bears a significant disadvantage, rendering their application in clinical testing currently impossible: NRBC cannot be expanded in culture, thereby ruling out metaphase chromosome analysis, an essential component of prenatal diagnosis. CD34+ cells do posses the potential for in vitro proliferation, however, they have been found to persist in the maternal circulation after delivery, thereby complicating diagnosis in consecutive pregnancies. Trophoblasts are not consistently detected in the maternal circulation. Moreover, due to the lack of a definitive fetal cell marker and a reliable sorting method, foolproof fetal cell identification of any of these cell types is not possible. This report outlines the obstacles that impede development of a method for noninvasive fetal cell sampling for prenatal genetic diagnosis, along with a description of our efforts to analyze simultaneously two fetal blood cell types, NRBC and CD34+ cells in maternal blood during pregnancy, and the problems encountered. This work and that of others lead us to suggest potential future directions to help develop this important technique.

Obsessive-compulsive disorder in patients with velocardiofacial (22q11 deletion) syndrome.

The study of neurogenetic microdeletion syndromes provides an insight into the developmental psychopathology of psychiatric disorders. The aim of the study was to evaluate the prevalence of psychiatric disorders, especially obsessive-compulsive disorder (OCD), in patients with velocardiofacial syndrome (VCFS), a 22q11 microdeletion syndrome. Forty-three subjects with VCFS of mean age 18.3 +/- 10.6 years were comprehensively assessed using semi-structured psychiatric interview and the Yale-Brown obsessive compulsive scale (Y-BOCS). Best estimate diagnoses were made on the basis of information gathered from subjects, parents, teachers, and social workers. Fourteen VCFS subjects (32.6%) met the DSM-IV criteria for OCD. OCD had an early age of onset and generally responded to fluoxetine treatment. It was not related to mental retardation. The most common obsessive-compulsive symptoms were contamination, aggression, somatic worries, hoarding, repetitive questions, and cleaning. Sixteen of the 43 patients (37.2%) had attention-deficit/hyperactivity disorder (ADHD), and 7 (16.2%) had psychotic disorder. The results of our study suggest that there is a strong association between VCFS and early-onset OCD. This finding may be significant in the understanding of the underlying genetic basis of OCD.

Familial vs sporadic ovarian tumors: characteristic genomic alterations analyzed by CGH.

OBJECTIVE: Our purpose was to get an overview of the genetic events leading to the development of familial and sporadic ovarian tumors and to identify chromosomal regions that may contain genes important in tumor progression. METHODS: The comparative genomic hybridization (CGH) technique was employed in a total of 46 epithelial ovarian or peritoneal tumors: 27 sporadic tumors, 11 tumors disected from BRCA1 mutation (185delAG) carriers, and eight from BRCA2 mutation (6174delT) carriers (familial tumors). RESULTS: The average number of genetic alterations (deletions and amplifications) was significantly (alpha=0.0069) higher in familial tumors (9.17 +/- 4.25 alterations per tumor in the BRCA1 mutation carriers and 7.25 +/- 6.06 in the BRCA2 mutation carriers) compared to the sporadic group (4.26 +/- 3.61 alterations per tumor). The pattern of the chromosome amplifications resembled in the three groups and the most common amplifications detected were at chromosomes 8q, 3q, and 2q. The pattern of the chromosomal deletions varied between the groups. Among the BRCA1 group, the most common deletions were in chromosomes 9 and 19. The BRCA2 group showed a lower frequency of deletions. Deletion of chromosome 16 and 22 were the most frequent ones. No specific chromosomal deletion was significantly indicated in the sporadic group. CONCLUSIONS: Familial ovarian tumors exhibit a significantly higher number of chromosomal aberrations and genomic imbalances and nonrandom genetic changes were characterized in the BRCA1 and BRCA2 groups.

True hermaphroditism with ambiguous genitalia due to a complicated mosaic karyotype: clinical features, cytogenetic findings, and literature review.

Abnormal recombination between the X and Y chromosomes during meiosis, occurring outside the pseudoautosomal region, can result in translocation of the SRY gene from the Y to the X chromosome, and consequently in abnormal sexual differentiation, such as the development of 46,XX males or true hermaphroditism. In this report we present clinical, cytogenetic, and molecular-cytogenetic data of a patient with ambiguous genitalia and true hermaphroditism, who had a unique mosaic karyotype, comprising three different cell lines: 46,XX(SRY+), 45,X(SRY+), and 45,X. The mosaic karyotype of our patient probably represents two different events: abnormal recombination between the X and Y chromosomes during paternal meiosis, and postzygotic loss of one of the X chromosomes. Replication studies demonstrated that in 80% of the XX cells, the SRY sequence was located on the active X chromosome. This finding suggests nonrandom X inactivation and, together with the presence of the SRY gene, explains the male phenotype of our patient. On the other hand, the presence of the 45,X cell line may have contributed to genital ambiguity. We conclude that fluorescence in situ hybridization (FISH) analysis with SRY probes is highly recommended and allows accurate diagnosis and optimal management in cases of 46,XX hermaphroditism and ambiguous genitalia.