Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study.

Numerical chromosome abnormalities were studied in single blastomeres from arrested or otherwise morphologically abnormal human preimplantation embryos. A 6-h FISH procedure with fluorochrome-labeled DNA probes was developed to determine numerical abnormalities of chromosomes X, Y, and 18. The three chromosomes were stained and detected simultaneously in 571 blastomeres from 131 embryos. Successful analysis including biopsy, fixation, and FISH analysis was achieved in 86.5% of all blastomeres. The procedure described here offers a reliable alternative to sexing of embryos by PCR and allows simultaneous ploidy assessment. For the three chromosomes tested, numerical aberrations were found in 56.5% of the embryos. Most abnormal embryos were polyploid or mosaics, and 6.1% were aneuploid for gonosomes or chromosome 18. Extrapolation of these results to all human chromosomes suggests that the majority of abnormally developing and arrested human embryos carry numerical chromosome abnormalities.     

Aneuploidy in Drosophila, I. Genetic test systems in the female Drosophila melanogaster for the rapid detection of chemically induced chromosome gain and chromosome loss

An account is provided of two genetic schemes in the Drosophila melanogaster female designed as rapid detectors of chemically induced aneuploidy, including both chromosome gain and chromosome loss. One scheme is referred to as FIX, in which the female carried free (heterozygously) inverted X (chromosomes) and the other, ZESTE, where females do not carry inversions and the X-linked sexually dimorphic zeste mutation plays the key role in the detection of aneuploid offspring. The principle attribute of the FIX system is that all euploid offspring are wild-type for body and eye color whereas aneuploid females have a yellow body and aneuploid males white eyes; int he ZESTE system all euploid individuals are wild-type for eye color, aneuploid females possess zeste-colored eyes and aneuploid males white eyes. In addition induced polyploidies (2X:2A gametes) appear as yellow and zeste male intersexes in the FIX and ZESTE systems, respectively. In this way all aneuploids are recognized immediately. Consequently, detection of compounds with weak effects requiring large sample sizes may be made in a fraction of the time associated with more traditional schemes for aneuploidy detection in Drosophila.     

Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens.

Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). We herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies.     

A retrospective study of preimplantation embryos diagnosed with monosomy by fluorescence in situ hybridization (FISH)

This report is a retrospective study of preimplantation embryos diagnosed with monosomy for chromosomes 13, 15, 16, 18, 21, 22, X and Y on day 3 to determine the rate of true positives, false positives and/or mosaicism and to assess if these embryos are suitable for in vitro fertilization (IVF) transfer. In a one year period, 80 patients went through preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Monosomy was diagnosed in 51 embryos. Fluorescence in situ hybridization (FISH) was then performed on the blastomeres at day 5-7 with commercially available probes using the same probe set that initially identified monosomy for chromosomes 13, 16, 21 and 22 or chromosomes 15, 18, X and Y. Based on FISH analysis, the monosomy diagnosed during routine PGD-AS analysis was confirmed in 17 of the 51 embryos. A euploid result for the specific chromosomes tested was observed in 16 of the 51 embryos while mosaicism was found in the remaining 18 embryos. This results in an estimated false positive rate of 3.8% for a diagnosis of monosomy. Reanalysis of these embryos demonstrates that the majority of monosomy diagnoses represents true monosomy or mosaicism and should be excluded for transfer in IVF. Furthermore, improved understanding from recent emerging data regarding the fate of oocytes in women with advanced maternal age undergoing IVF to the development of early embryos may provide a valuable insight into the mechanism of chromosome mosaicism.     

Prenatal detection of chromosome aneuploidies in uncultured chorionic villus samples by FISH.

We developed a 1-d FISH assay for detection of numerical chromosome abnormalities in uncultured chorionic villus samples (CVS). Probes specific for chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. Aneuploidy detection using this assay was directly compared with the results obtained by conventional cytogenetic analysis in a consecutive, clinical study of 2,709 CVS and placental samples. The FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. On the basis of these results, we generated FISH-assay cutoff values that discriminated between karyotypically normal and aneuploid samples. Samples with mosaicism and a single sample with possible heritable small chromosome X probe target were exceptions and showed poor agreement between FISH results and conventional cytogenetics. We conclude that the FISH assay may act as a more accurate and less labor-demanding alternative to "direct" CVS analysis.     

A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo

Aneuploid embryos diagnosed by FISH-based preimplantation genetic screening (PGS) have been shown to yield euploid lines of human embryonic stem cells (hESCs) with a relatively high frequency. Given that the diagnostic procedure is usually based on the analysis of 1–2 blastomeres of 5 to 10-cell cleavage-stage embryos, mosaicism has been a likely explanation for the phenomena. However, FISH-based PGS can have a significant rate of misdiagnosis, and therefore some of those lines may have been derived from euploid embryos misdiagnosed as aneuploid. More recently, coupling of trophectoderm (TE) biopsy at the blastocyst stage and array-CGH lead to a more informative form of PGS. Here we describe the establishment of a new line of hESCs from an embryo with a 43,XX,dup(9q),+12,-14,-15,-18,-21 chromosomal content based on array-CGH of TE biopsy. We show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo´s missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the TE lineage.     

Oligonucleotide Arrays vs. Metaphase-Comparative Genomic Hybridisation and BAC Arrays for Single-Cell Analysis: First Applications to Preimplantation Genetic Diagnosis for Robertsonian Translocation Carriers

Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.     

Editor's choice: Correction of Down syndrome and Edwards syndrome aneuploidies in human cell cultures

Aneuploidy, an abnormal number of chromosomes, has previously been considered irremediable. Here, we report findings that euploid cells increased among cultured aneuploid cells after exposure to the protein ZSCAN4, encoded by a mammalian-specific gene that is ordinarily expressed in preimplantation embryos and occasionally in stem cells. For footprint-free delivery of ZSCAN4 to cells, we developed ZSCAN4 synthetic mRNAs and Sendai virus vectors that encode human ZSCAN4. Applying the ZSCAN4 biologics to established cultures of mouse embryonic stem cells, most of which had become aneuploid and polyploid, dramatically increased the number of euploid cells within a few days. We then tested the biologics on non-immortalized primary human fibroblast cells derived from four individuals with Down syndrome—the most frequent autosomal trisomy of chromosome 21. Within weeks after ZSCAN4 application to the cells in culture, fluorescent in situ hybridization with a chromosome 21-specific probe detected the emergence of up to 24% of cells with only two rather than three copies. High-resolution G-banded chromosomes further showed up to 40% of cells with a normal karyotype. These findings were confirmed by whole-exome sequencing. Similar results were obtained for cells with the trisomy 18 of Edwards syndrome. Thus a direct, efficient correction of aneuploidy in human fibroblast cells seems possible in vitro using human ZSCAN4.     

Preimplantation genetic diagnosis--an overview.

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.     

Microdensitometric evaluation of the DNA content,as ploidy parameter,of spermatozoa in the polymorphic chromosomal system of Akodon molinae cabrera (rodentia,cricetidae)

The influence of chromosome variability on the production of euploid spermatozoa was investigated in a suitable biological model, the Akodon molinae system. This consists of individuals whose chromosome constitution is 2n = 42, 2n = 43, or 2n = 44. The only difference between these three karyotypes occurs through a Robertsonian rearrangement combined with two pericentric inversions. Thus, the animals with 2n = 42 (simple homozygotes or SH) have two large metacentric chromosomes number 1; animals with 2n = 43 (heterozygotes or Ht) have a chromosome 1 and two subterminal chromosomes la and lb homologues of the long and short arms of the chromosome 1, respectively; animals with 2n = 44 (double homozygotes or DH) have a pair of la and a pair of lb chromosomes. The gametic euploidy frequency correlated with each chromosome constitution was evaluated on the basis of the DNA content of spermatozoa, which was determined microdensitometrically after the Feulgen reaction, taking into account the site of the spermatozoa along the male genital tract. A comparative assessment of gametic aneuploidy frequency in caput epididymis versus vas deferens demonstrated (1) a falloff in euploid production in passing from the 2n = 42 to the 2n = 44 chromosome forms, alongside a high degree of intragroup variability, and (2) a lower aneuploidy frequency in the vas deferens than in caput epididymis in all the forms considered. These two features, taken together with similar results in the mouse chromosome variability system, suggest that a selection mechanism is operative against aneuploid spermatozoa in the epididymis. This finding is of interest in a wider perspective, since it might turn out to be valid for many mammals.     

Cytological and breeding behavior of pentaploids derived from 3x × 4x crosses in potato

Cytology and breeding behavior of Solanum commersonii - S. tuberosum hybrids derived from 3 x x 4 x crosses was examined. The chromosome number of hybrids ranged from hypo-pentaploid (2 n=5 x - 8=52), to hyper-pentaploid (2 n=5 x + 7=67), with the euploid pentaploid 2 n=5 x=60 class predominant. The high variability in chromosome number of the 3 x x 4 x hybrids was attributed to the fact that meiotic restitution during megasporogenesis of the 3 x female may have involved poles with various chromosome numbers, resulting in 2 n eggs with 24-48 chromosomes. Microsporogenesis analyses provided evidence that chromosome pairing between S. commersonii and S. tuberosum genomes occurred. In addition, chromosome distribution at anaphase I and anaphase II revealed an average chromosome number of 29.5 and 29.1 per pole, respectively. To further study the extent of transmission of extra genome chromosomes from pentaploids, 5 x x 4 x and 4 x x 5 x crosses were performed, and the chromosome number of resulting progeny was determined. Ploidy ranged from 2 n=4 x=48 to 2 n=5 x=60 following 5 x x 4 x crosses, and from 2 n=4 x + 1=49 to 2 n=5 x=60 following 4 x x 5 x crosses. These results provided indirect evidence that the pentaploid hybrids produced viable aneuploid gametes with a chromosome number ranging from 24 to 36. They also demonstrated that gametes with large numbers of extra chromosomes can be functional, resulting in sporophytes between the 4 x and 5 x ploidy level. Fertility parameters of crosses involving various (aneuploid) pentaploid genotypes were not influenced by chromosome number, suggesting a buffering effect of polyploidy on aneuploidy. The possibility of successfully using (aneuploid) pentaploid genotypes for further breeding efforts is discussed.     

Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.     

Increased Frequency of Aneuploidy in Long-Lived Spermatozoa

Aneuploidy commonly causes spontaneous abortions, stillbirths, and aneuploid births in humans. Notably, the majority of sex chromosome aneuploidies in live births have a paternal origin. An increased frequency of aneuploidy is also associated with male infertility. However, the dynamics and behavior of aneuploid spermatozoa during fertilization in humans have not been studied in detail. Therefore, we compared the frequency of aneuploidy and euploidy in live spermatozoa from normozoospermic men over a 3-day period. To assess the dynamics and behavior of aneuploid spermatozoa, we simultaneously evaluated sperm viability using the hypo-osmotic swelling test and sperm aneuploidy using fluorescence in situ hybridization. Whereas the frequency of viable euploid spermatozoa significantly decreased over 3 days, the frequency of viable spermatozoa with aneuploidy interestingly showed a time-dependent increase. In addition, spermatozoa with abnormal sex chromosomes survived longer. To compared with spermatozoa with other swelling patterns, those with tail-tip swelling patterns had a lower frequency of aneuploidy at all time points. This study revealed the novel finding that the frequency of aneuploid spermatozoa with fertilization capability significantly increased compared to that of euploid spermatozoa over 3 days, suggesting that aneuploid spermatozoa can survive longer than euploid spermatozoa and have a greater chance of fertilizing oocytes.     

DNA content of wheat monosomics at interphase estimated by flow cytometry

 Two complete, independently maintained sets of 21 monosomic wheat lines derived from cv. ‘Chinese Spring’ were analyzed for their DNA content at the G1 stage with flow cytometry. The DNA content of individual chromosomes was estimated by subtracting the value of a monosomic line from that of euploid wheat. Our data show that the estimated 2C DNA of individual wheat chromosomes in 21 monosomics at the G1 stage ranges from about 0.58 pg in chromosome 1D to approximately 1.12 pg in chromosome 3A. The A genome (2C=6.15 pg) seems to contain more DNA than the B (2C=6.09 pg) and D (2C=5.05 pg) genomes. Analysis of variance showed significant differences (α=0.01) in DNA content both among homoeologous groups and among genomes. Our estimates of interphase DNA content of wheat chromosomes from monosomic lines were poorly correlated to the chromosome sizes at metaphase (r=0.622, P≤0.01). This poor correlation might be due to differential coiling among chromosomes during cell division, possible bias of fluorochrome binding to heterochromatin, or heterogeneity among monosomic lines. Finally, flow cytometry may aid but cannot replace cytological checks in aneuploid maintenance. Received: 21 January 1997 / Accepted: 23 June 1997     

Chromosomal composition of aneuploid clones with different DNA contents in head and neck squamous cell carcinomas as determined by combined flow cytometry and fluorescence in situ hybridization.

Studies with DNA flow cytometry (FCM) have shown that DNA contents of aneuploid tumour clones vary in a wide range. The aim of this study was to analyse whether homologous chromosomal changes exist despite the individual differences that may be of general relevance for the development of gross aneuploidy in squamous cell carcinomas of the head and neck. Fluorescence in situ hybridization (FISH) with 13 centromere-specific DNA probes was applied to 3 diploid and 11 aneuploid tumours with DNA indices ranging between 0.8 and 2.2. Disomic and monosomic cell populations were prevalent findings in DNA-diploid tumours. Polysomies were common in aneuploid tumours. Different degrees of aneusomy for identical chromosomes were recurrent features in aneuploid tumours. FISH signal heterogeneity was identified for all chromosomes. The mean number of aneusomic cell populations identified for DNA-aneuploid tumours ranged between 1.6 for chromosome 17 and 3.1 for chromosome 3. Inconsistencies between FISH and FCM data may indicate that centromere-specific DNA probes identify gains and losses of marker DNA due to complex karyotypic rearrangements rather than absolute changes in chromosome numbers. Overall, there was no evidence of the critical involvement of particular chromosomes in the development of different DNA contents.     

Experimental alterations in the chromosome constitution of Sciara

Summary Using two reciprocal translocations between chromosomes X and IV in S. coprophila it has been possible to derive two kinds of aneuploid females. Both of the aneuploid complements are detrimental — one is lethal, the other may give rise to viable, fertile adults. Males with aneuploid somatic complements have not been obtained; three different aneuploid complements were tested but gave negative results. Males with euploid soma and aneuploid germ line have been produced in three separate instances; they are viable and fertile.Dedicated to Professor H. Bauer on the occasion of his sixtieth birthday.The studies reported here were supported by grant GB-42 from the National Science Foundation.     

Phenotypic Variation and Changes of Chromosome Number in Plantlets Regenerated from Potato Protoplasts

Morphological variation and change of chromosome number in the plantlets of potato (Solanum tuberosum L. cv. Xiao Yie Zi × Duo Zi Bai) regenerated from mesophyll protoplasts have been studied. The normal plantlets from protoplasts were similar to parent plants. Their chromosome numbers were 2n = 48±or 2n= 72 of euploid. The plantlets with distinctive phenotypic variation were likely to be aneuploid with increased chromosome numbers.     

Interchromosomal exchange of a large subtelomeric segment in a Plasmodium falciparum cross.

Duplications and interchromosomal transpositions of chromosome segments are implicated in the genetic variability of Plasmodium falciparum malaria parasites. One parasite clone, HB3, was shown to lack a subtelomeric region of chromosome 13 that normally carries a PfHRPIII gene. We show here that the chromosome 13 segment carrying PfHRPIII was replaced in HB3 by a duplicated terminal segment from chromosome 11. Mapping results indicate that the segment includes at least 100-200 kb of subtelomeric DNA and contains duplicated copies of the Pf332 and RESA-2 genes. We followed inheritance of this duplication in a genetic cross between the HB3 and another P.falciparum clone, Dd2, that is euploid for the Pf332, RESA-2 and PfHRPIII genes. Three types of progeny from the cross showed expected inheritance forms: a Dd2 euploid parent type, an HB3 aneuploid parent type, and a recombinant euploid type that carried PfHRPIII from Dd2 chromosome 13 and Pf332 from HB3 chromosome 11. However, a fourth euploid progeny type was also observed, in which the chromosome 13 segment from HB3 was transposed back to replace the terminus of chromosome 11. Three of 14 individual progeny were of this type. These findings suggest a mechanism of recombination from subtelomeric pairing and exchange between non-homologous chromosomes in meiosis.     

Proliferative advantage of specific aneuploid cells drives evolution of tumor karyotypes

Most tumors have abnormal karyotypes, which arise from mistakes during mitotic division of healthy euploid cells and evolve through numerous complex mechanisms. In a recent mouse model with increased chromosome missegregation, chromosome gains dominate over losses both in pretumor and tumor tissues, whereas T-cell lymphomas are characterized by gains of chromosomes 14 and 15. However, the quantitative understanding of clonal selection leading to tumor karyotype evolution remains unknown. Here we show, by introducing a mathematical model based on a concept of a macro-karyotype, that tumor karyotypes can be explained by proliferation-driven evolution of aneuploid cells. In pretumor cells, increased apoptosis and slower proliferation of cells with monosomies lead to predominant chromosome gains over losses. Tumor karyotypes with gain of one chromosome can be explained by karyotype-dependent proliferation, whereas, for those with two chromosomes, an interplay with karyotype-dependent apoptosis is an additional possible pathway. Thus, evolution of tumor-specific karyotypes requires proliferative advantage of specific aneuploid karyotypes.