The peptide nucleic acids: a new way for chromosomal investigation on isolated cells?

The development of nucleic acid analogues has become an important feature due to the potential use of this new biomolecular tool in genetic diagnostics and investigations. Among all the synthetic oligonucleotides designed, the peptide nucleic acids (PNA) constitute a remarkable class of nucleic acid mimics, with important physico-chemical properties which have been exploited to develop a wide range of powerful biomolecular tools, including molecular probes, biosensors and anti-gene agents. New applications of PNA involve their use as hybridization probes, and consequently the PNA technology is now developing within the field of in situ hybridization techniques. Recent studies have reported the successful use of centromeric PNA probes on human lymphocytes, sperm as well as on isolated oocytes and blastomeres. Muticolour PNA protocols have been described for the specific identification of several human chromosomes. These data show that PNA could become a powerful complement to fluorescence in situ hybridization (FISH) for in situ chromosomal investigation, especially on isolated cells. The present paper gives an overview of the properties of PNA and the assays exploiting PNA technology in molecular genetics and cytogenetics.     

Sequential FISH analysis using competitive displacement of labelled peptide nucleic acid probes for eight chromosomes in human blastomeres

BACKGROUND: The aim was to introduce a new strategy based on peptide nucleic acid (PNA) probes and competitive displacement for using fluorescence in-situ hybridization (FISH) analysis on human blastomeres. METHODS: Sequential FISH analysis with PNA probes and competitive displacement was performed using three different probe sets. The first set consisted of labelled probe only. The second and third sets included labelled as well as unlabelled probe, corresponding to the labelled probes in the previous cycles. The probes for enumeration were for chromosome 1, 13, 16, 17, 18, 21, X and Y. RESULTS: The performance of PNA probes was similar to the established DNA probes. The strategy of competitive displacement resulted in a destabilization of already bound probe before the next FISH cycle at only 50 degrees C, which allowed for up to five sequential FISH cycles without loss of signal. CONCLUSIONS: PNA probes are a good alternative to DNA probes in the present set-up, since the low temperature required both for binding and destabilization of PNA probes minimizes the loss of signal, and several FISH cycles can therefore be carried out before FISH errors occur.     

PNA on human sperm: a new approach for in situ aneuploidy estimation

Peptide nucleic acids (PNAs) are a relatively new class of synthetic DNA mimics based on a peptide-like backbone. Since their introduction, PNA probes have become established as an efficient variation on the standard FISH procedure for chromosomal identification. In this report we have experimented with centromeric PNA probes on human sperm preparations. Both NaOH and DTT sperm decondensation procedures have been tested and comparative estimates of disomies X, Y and 1 have been performed in sperm from two donors using PNA, FISH and PRINS techniques. Similar results were obtained with the three methods, demonstrating the efficiency of PNA probes in the analysis of human sperm. The fast kinetics, stability and high specificity of PNA probes make PNA-based methodologies very valuable for in situ cytogenetic investigations.     

Differentiation of Candida albicans and Candida dubliniensis by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes

The recent discovery of Candida dubliniensis as a separate species that traditionally has been identified as Candida albicans has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection and identification of microorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridization characteristics, and the neutral backbone of PNA probes offers significant advantages in whole-cell in situ hybridization assays. In this study, we developed PNA probes targeting the rRNAs of C. albicans and C. dubliniensis and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between C. albicans and C. dubliniensis. Liquid cultures were smeared onto microscope slides, heat fixed, and then hybridized for 30 min. Unhybridized PNA probe was removed by washing, and smears were examined by fluorescence microscopy. Evaluation of the PNA FISH method using smears of 79 C. dubliniensis and 70 C. albicans strains showed 100% sensitivity and 100% specificity for both PNA probes. We concluded that PNA FISH is a powerful tool for the differentiation of C. albicans and C. dubliniensis.     

Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes

Chromosomal aberrations are the major cause of pre- and post- implantation embryo wastage and some studies suggest that half of all human conception have a chromosomal abnormality. Analysis of gametes provides information on the origin of these chromosomal aberrations. The purpose of this study was to develop a reliable multi-probe fluorescence in-situ hybridization (FISH) procedure that would enable us to investigate aneuploidy in unfertilized oocytes subjected to intracytoplasmic sperm injection (ICSI). Oocytes were spread with HCl and Tween 20 solution, and then two rounds of triple-probe FISH were performed on each oocyte using directly-labelled centromeric probes: chromosomes 1, 7, 15 (overnight hybridization); chromosomes 1, X, Y (2 h hybridization). After the first round, the slides were counterstained and evaluated, and the positions of FISH signals were recorded. For the second round, the counterstain was removed and the second probe cocktail was applied. The chromosome 1 probe was an internal control for the two hybridization procedures, while the Y chromosome probe was used to detect sperm DNA. To evaluate the method, a total of 79 oocytes from 27 patients were studied. Of these, 67 (84.8%) were successfully spread and 97% of these oocytes exhibited discernible FISH signals. Upon lysis, oocytes exhibited one or more DNA fragments (mean 1.9, range 1-3). Of the 65 analysable oocytes, 17 (26.2%) displayed a normal haploid chromosome constitution with paired spots for the two chromatids. A further 23 oocytes (35.4%) showed an ambiguous chromosome complement due to an abnormal number of DNA fragments which may have resulted from loss of DNA during spreading or to an abnormal oocyte, while 25 oocytes (38.4%) displayed aneuploidy for one or more of the chromosomes studied. In conclusion, this new approach is a quick and efficient method with which numerical chromosomal abnormalities in human oocytes can be studied; interpretation of the patterns of DNA fragments and FISH signals requires further clarification.      

Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization

Chromosomal rearrangements are frequently monitored by fluorescence in situ hybridization (FISH) using large, recombinant DNA probes consisting of contiguous genomic intervals that are often distant from disease loci. We developed smaller, targeted, single-copy probes directly from the human genome sequence. These single-copy FISH (scFISH) probes were designed by computational sequence analysis of approximately 100-kb genomic sequences. ScFISH probes are produced by long PCR, then purified, labeled, and hybridized individually or in combination to human chromosomes. Preannealing or blocking with unlabeled, repetitive DNA is unnecessary, as scFISH probes lack repetitive DNA sequences. The hybridization results are analogous to conventional FISH, except that shorter probes can be readily visualized. Combinations of probes from the same region gave single hybridization signals on metaphase chromosomes. ScFISH probes are produced directly from genomic DNA, and thus more quickly than by recombinant DNA techniques. We developed single-copy probes for three chromosomal regions-the CDC2L1 (chromosome 1p36), MAGEL2 (chromosome 15q11.2), and HIRA (chromosome 22q11.2) genes-and show their utility for FISH. The smallest probe tested was 2290 bp in length. To assess the potential utility of scFISH for high-resolution analysis, we determined chromosomal distributions of such probes. Single-copy intervals of this length or greater are separated by an average of 29.2 and 22.3 kb on chromosomes 21 and 22, respectively. This indicates that abnormalities seen on metaphase chromosomes could be characterized with scFISH probes at a resolution greater than previously possible.     

FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology

BACKGROUND: The data are compiled from two multicentre, prospectively randomized studies on the effect of follicular fluid meiosis-activating sterol (FF-MAS) on human oocytes. The donated oocytes were exposed either to test doses of FF-MAS or to control solutions. The data from the control groups are presented with chromosomal status of the embryos correlated to embryo morphology. METHODS: The study includes 144 randomly selected donated human oocytes. The nucleus from each blastomere was fixed separately and fluorescence in-situ hybridization (FISH) using seven probes (13, 16, 18, 21, 22, X and Y) was performed. RESULTS: Analysis of 103 pre-embryos containing 479 blastomeres resulted in 424 blastomeres with clear FISH signals. Of these blastomeres, 55% were normal diploid and 45% were abnormal. At a pre-embryonic level, 53% were classified as normal containing >or=50% normal blastomeres while 31% of the pre-embryos were classified as uniformly normal. Abnormality rate was significantly increased in the pre-embryos with unevenly sized blastomeres and with increasing degree of fragmentation at 68 h after fertilization. Applying criteria for good embryo quality significantly increased the rate of chromosomally normal pre-embryos from 53 to 75%. CONCLUSIONS: The data demonstrate the high degree of genetic heterogeneity in a randomly selected pool of donated pre-embryos from an IVF programme. Further, we found that uniformity of blastomere size, degree of fragmentation and cleavage kinetics reflect the cytogenetic status of the pre-embryo and are therefore important in the selection of pre-embryos.     

Molecular cytogenetic studies of chromosomal abnormalities and disorders in nervous and mental diseases: search for biological markers for diagnosis

Molecular cytogenetic and cytogenetic studies of chromosomal disorders in patients with nervous and mental disorders were conducted using currently available approaches, including fluorescence in situ hybridization (FISH) with an original collection of centromeric, telomeric, region-specific DNA probes. A novel in situ hybridization protocol for rapid (15-30 min) chromosomal detection procedure and directly fluoresceinated DNA probes are recommended for use in mental retardation and congenital malformations. Chromosomal abnormalities could be detected in postnatal cases with chromosomal structural rearrangements, aneuplodies of gonosomes (including mosaicisms) and autosomes (including marker chromosomes). Molecular cytogenetics (or FISH diagnosis) can be used when classical cytogenetic methods are insufficient. The authors' experience shows that FISH should be utilized only as an adjunctive test for classical cytogenetic studies when banding techniques are ineffective; cytogenetic methods should be utilized for the preclinical diagnosis of Rett's syndrome. Detection by original probes gives an additional possibility in FISH analysis. Molecular cytogenetic methods are shown to provide a rapid accurate approach to studying and diagnosing chromosomal anomalies and disorders in mental retardation with congenital malformations and Rett's syndrome in children and to exploring aneuploidies in the postmortem cell samples from schizophrenic patients.     

Karyotyping of human oocytes by cenM-FISH, a new 24-colour centromere-specific technique

BACKGROUND: Metaphase II (MII) chromosome complements are difficult to karyotype. The objective of this study was to investigate the efficiency and limitations of centromere-specific multiplex fluorescence in situ hybridization (cenM-FISH), a new 24 colour FISH technique using centromere-specific probes, to analyse the whole chromosome complement within human oocytes. METHODS: Oocytes were donated by 34 patients undergoing ovarian stimulation and IVF. The MII oocytes were analysed by means of cenM-FISH, while the confirmation of results was performed by FISH and/or by analysing the corresponding first polar bodies using comparative genomic hybridization (CGH). RESULTS: A total of 30 cells, corresponding to 16 oocytes and 14 first polar bodies, were successfully karyotyped by either cenM-FISH or CGH. The incidence of aneuploidy was 25%, and eight out of nine aneuploidy events were confirmed by CGH and FISH. CONCLUSIONS: We demonstrate here for the first time that the identification of any numerical abnormality in oocytes is feasible using cenM-FISH. Despite the fact that the fixation efficiency remains low, the present results confirm the advantage of analysing the whole set of chromosomes to make an accurate estimation of the aneuploidy rate in human oocytes.     

Feasibility study of repeated fluorescent in-situ hybridization in the same human blastomeres for preimplantation genetic diagnosis

In order to increase the number of chromosomes examined in each blastomere, we have developed a repeated fluorescent in-situ hybridization (FISH) procedure by which six or more chromosomes can be analysed per blastomere of a human embryo. Three consecutive FISH procedures with directly-labelled fluorescent Vysis DNA probes were carried out for examination of chromosomes X, Y, 11, 13, 18 and 21 in the same blastomeres (n = 126) and lymphocytes (n = 164). Based on the initial number of nuclei, the percentages of nuclear loss and presence of signals were 3 and 92% respectively in blastomeres; 6 and 91% respectively in lymphocytes after the first FISH; 7 and 87% respectively in blastomeres and 10 and 86% respectively in lymphocytes, after the second FISH. These percentages were 13 and 78% respectively in blastomeres and 14 and 81% respectively in lymphocytes after the third FISH. The FISH procedure was repeated successfully in a couple for preimplantation genetic diagnosis of chromosomal aneuploidies in biopsied blastomeres of their embryos in our clinic. In conclusion, it is feasible to carry out repeated FISH procedures in the same blastomeres. Six or more chromosomes of a single blastomere may be examined using this procedure.      

Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF

BACKGROUND: In order to assess the frequency of aneuploidy and mosaicism in embryos obtained from IVF patients aged <38 years, preimplantation genetic screening (PGS) was performed after biopsy of two blastomeres. Furthermore, the reliability of this diagnosis was assessed by performing reanalysis of the embryo on day 5. METHOD: The copy numbers of 10 chromosomes (1, 7, 13, 15, 16, 18, 21, 22, X and Y) were investigated by fluorescence in situ hybridization (FISH) analysis. Embryos that were found to be abnormal or of insufficient morphological quality were cultured until day 5 and reanalysed. Results obtained were compared to the day 3 blastomere analysis. RESULTS: After analysis of 196 embryos (one cell in 38% and two cells in 62%), only 36% of the embryos were found to be normal on day 3. After analysis of two blastomeres, 50% showed chromosomal mosaicism. Comparison of the FISH results from day 3 blastomeres and day 5 embryos yielded an overall cytogenetic confirmation rate of 54%. CONCLUSIONS: The rates of mosaicism and aneuploidy in these embryos from young IVF patients are similar to those published for older women. We found the best confirmation rate after a diagnosis based on two cells, where both blastomeres showed the same chromosomal abnormality. In contrast, after a mosaic diagnosis the confirmation rate was low. The present study provides the first detailed reanalysis data of embryos analysed by PGS and clearly demonstrates the impact of mosaicism on the reliability of the PGS diagnosis.     

Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting

The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed a precise in-situ identification of both chromosomes and free chromatids, and consequently a reliable analysis of chromosomal segregation errors. Of the 104 analysed oocytes, 84 (80.7%) displayed a normal chromosome constitution. Three cases of chromosome non-disjunction (2.8%) were found, whereas seven oocytes (6.7%) presented extra single chromatids. In addition, 12 oocytes (11.5%) showed balanced pre-division of one pair of sister chromatids. Although this phenomenon was not classified as aneuploidy, it could lead to aneuploidy at anaphase II. Abnormalities were observed in all the targetted chromosomes. The present data confirm that both whole chromosome non-disjunction and premature chromatid separation constitute the two major mechanisms of aneuploidy in human female meiosis.     

Carrier-specific breakpoint-spanning DNA probes: an approach to preimplantation genetic diagnosis in interphase cells

Carriers of chromosomal inversions or other balanced rearrangements represent a significant fraction of patients in in-vitro fertilization (IVF) programmes due to recurrent reproductive problems. In most cases, chromosomal imbalance in fertilized oocytes is incompatible with embryo survival leading to increased rates of spontaneous abortions. Assuming that a fraction of the germ cells is karyotypically normal, these patients would greatly benefit from efficient procedures for generation and use of breakpoint-specific DNA hybridization probes in preconception and preimplantation genetic diagnosis (PGD). We describe the generation of such patient-specific probes to discriminate between normal and aberrant chromosomes in interphase cells. First, a large insert DNA library was screened for probes that bind adjacent to the chromosomal breakpoints or span them. Then, probe and hybridization parameters were optimized using white blood cells from the carrier to increase in hybridization signal intensity and contrast. Finally, the probes were tested on target cells (typically polar bodies or blastomeres) and a decision about the colour labelling scheme was made, before the probes can be used for preconception or preimplantation genetic analysis. Thus, it was demonstrated that cells with known structural abnormalities could be detected, based on hybridization of breakpoint spanning yeast artificial chromosome (YAC) DNA probes in interphase cells.      

Clinical applications of two-color telomeric fluorescence in situ hybridization for prenatal diagnosis: Identification of chromosomal translocation in five families with recurrent miscarriages or a child with multiple congenital anomalies

Two-color fluorescence in situ hybridization (FISH) analysis using human chromosome arm-specific telomeric probes (telomeric probes) was used successfully to detect each derivative chromosome of a translocation carrier in five couples who requested a prenatal diagnosis in future pregnancies. Most of the human chromosome terminal bands are G-band-negative, and even FISH analysis using whole-chromosome painting (wcp) probes are often of insufficient complexity to detect subtle chromosomal changes. A complete set of human telomeric probes was developed to improve the sensitivity of diagnosis of microdeletions or other cryptic rearrangements in telomeric regions. Two-color telomeric FISH was the only possible method for precise prenatal diagnosis of one of the couples, because the carrier's chromosomal aberration was too subtle to be detected by wcp FISH or conventional methods. We have demonstrated that two-color telomeric FISH has the potential to be a powerful new tool in the detection of cryptic chromosomal rearrangements involving telomeric regions in prenatal diagnosis precisely and in time. Received: September 9, 1998 / Accepted: October 23, 1998     

Multicolor FISH in two and three dimensions for clastogenic analyses

Chemicals may induce both numerical and structural aberrations. In addition to these chromosomal mutations, chemicals may render cells genetically unstable, which may result in chromosomal instability. For a detailed analysis, sophisticated approaches at single cell resolution are needed. Such approaches have become feasible by recent developments in molecular cytogenetics. In particular, new multicolor fluorescence in situ hybridization (FISH) technologies allow us now to study the effects of chemicals on chromosomes with unprecedented resolution. FISH provides opportunities to analyze the genome in two dimensions, i.e. on metaphase spreads, or in three dimensions, i.e. in interphase nuclei. An arsenal of diverse multicolor FISH approaches has been developed, which allows the analysis of the entire genome with one hybridization on metaphase spreads or the detailed visualization of selected chromosomal regions within intact interphase nuclei. These developments have been complemented by new resources for DNA probes, which have evolved from the human genome project. Here we will review the latest developments and provide some examples in which multicolor FISH technologies were applied to elucidate the effect of chemicals on chromosomes.     

Comparative genomic hybridization analysis of human oocytes and polar bodies

BACKGROUND: Classical cytogenetic methods and fluorescent in situ hybridization (FISH) have been employed for the analysis of chromosomal abnormalities in human oocytes. However, these methods are limited by the need to spread the sample on a microscope slide, a process that risks artefactual chromosome loss. Comparative genomic hybridization (CGH) is a DNA-based method that enables the investigation of the entire chromosome complement. We optimized and evaluated a CGH protocol for the chromosomal analysis of first polar bodies (PBs) and oocytes. The protocol was then employed to obtain a detailed picture of meiosis I errors in human oogenesis. METHODS: 107 MII oocyte-PB complexes were examined using whole genome amplification (WGA) and CGH. RESULTS: Data was obtained for 100 complexes, donated from 46 patients of average age 32.5 (range 18-42). 22 complexes from 15 patients were abnormal, giving an aneuploidy rate of 22%. CONCLUSIONS: The results presented in this study more than double the quantity of CGH data from female gametes currently available. Abnormalities caused by whole chromosome non-disjunction, unbalanced chromatid predivision and chromosome breakage were reliably identified using the CGH protocol. Analysis of the data revealed a preferential participation of chromosome X and the smaller autosomes in aneuploidy and provided further evidence for the existence of age-independent factors in female aneuploidy.     

The combination of polar body and embryo biopsy does not affect embryo viability

BACKGROUND: The biopsy of both polar bodies and a blastomere from the same embryo was investigated as an approach aimed at increasing the quantity of DNA available for genetic analysis in preimplantation embryos. METHODS: In 113 cycles, preimplantation genetic diagnosis (PGD) was performed for aneuploidy: 19 cycles underwent polar body biopsy, 32 cycles had both polar body and blastomere biopsy done, and the remaining 62 cycles underwent blastomere biopsy. The chromosomal analysis was performed in a two-round fluorescence in situ hybridization (FISH) protocol with probes specific for the chromosomes X, Y, 13, 15, 16, 18, 21 and 22. RESULTS: The morphological evaluation of the analysed embryos demonstrated similar rates of development irrespective of the biopsy procedure. Accordingly, the implantation rate did not differ significantly in the three biopsy groups and was 15% after polar body biopsy, 26% after the combined biopsy procedures of polar bodies and blastomeres, and 25% after blastomere biopsy. CONCLUSIONS: The removal of a blastomere subsequent to polar body biopsy does not seem to have negative effects on embryo viability. This approach could be especially valuable for a combined diagnosis of aneuploidy and single-gene disorders in preimplantation embryos generated by couples at high reproductive risk.     

Rapid identification of Staphylococcus aureus in blood cultures by a combination of fluorescence in situ hybridization using peptide nucleic acid probes and flow cytometry

Fluorescence in situ hybridization (FISH) using peptide nucleic acid probes (PNAs) allows the identification of Staphylococcus aureus from human blood culture samples. We present data revealing that the combination of PNA FISH and flow cytometry is a possible approach for the noncultural identification of staphylococci in blood cultures.     

Generation of Chromosome Painting Probes from Single Chromosomes by Laser Microdissection and Linker-Adaptor PCR

Fluorescence in situ hybridization (FISH) plays an essential role in research and clinical diagnostics. The versatility and resolution of FISH depends critically on the probe set used. Here, we describe an improved approach for the generation of specific DNA probes from single copies of chromosomes. Single chromosomes or single chromosomal regions were microdissected by laser pressure catapulting and amplified using linker-adaptor PCR. The probes were labeled and tested in various scenarios including multicolor-FISH experiments employing up to seven different fluorochromes. FISH confirmed the specific and even staining of the respective chromosomal regions. Furthermore, the capability of these probes to detect even small translocations (<3 Mb) suggests that the dissected regions are completely represented in the generated painting probes.     

Development of a non-human primate sperm aneuploidy assay tested in the rhesus macaque (Macaca mulatta)

Numerical chromosome aberrations in germ cells are important factors contributing to abnormal reproductive outcomes. Fluorescence in situ hybridization onto spermatozoa (sperm-FISH) has allowed the study of the influence of a wide range of biological factors and chemical exposure on aneuploidy incidences in human sperm as well as in mouse and rat animal models. The assay presented here extends the applicability of the sperm-FISH method to non-human primates and was tested in the prevalent model species, the rhesus macaque. The assay provides probes for macaque chromosomes 17, 18, 19, 20, X and Y, the homologues of human chromosomes 13, 18, 19, 16, X and Y, respectively. The analysis of 11 000 spermatozoa each from five individuals revealed spontaneous sex chromosomal disomy frequencies (X: 0.08%; Y: 0.09%) and an average autosomal disomy frequency (0.03%) coinciding with some of the lowest incidences scored in human studies. The non-human primate sperm-FISH assay provides a fast and efficient tool complementing the available analysis methods in non-human primate exposure studies. Since the assay employs large locus-specific FISH probes representing evolutionary conserved DNA sequences, it can be expected that the assay is also applicable to other cercopithecoid and hominoid non-human primate species.