The chromosome constitution of human preimplantation embryos fertilized in vitro

The chromosome constitution of five haploid, 178 diploid and11 triploid embryos fertilized in vitro was determined afterfixation on day 2 or day 3 of development. Karyotype analysisof 178 diploid embryos revealed abnormalities in 40 (22.5%)cases: 34 (19.1%) aneuploids, four (2.2%) mosaic embryos andtwo (1.1%) structural anomalies were identified. The majorityof aneuploid karyotypes (28/34) involved a single chromosomebut six embryos had aneuploidy of two or three chromosomes.The E group was most frequently involved in aneuploid karyotypes(10/23 hyperdiploid embryos) and trisomy 16, the most commonsingle anomaly in diploid embryos, was detected in 2.2% (4/178)of cases. Only one case of sex chromosome monosomy was identified.An excess of female karyotypes was detected in abnormal cases(sex ratio 0.48); this ratio was significantly (p< 0.05)different from that observed in normal cases (74: 64, XY: XX).The incidence of aneuploidy increased with maternal age butthis did not reach statistical significance. Embryo morphologyand growth rate, assessed by embryo development rating (EDR),did not distinguish between normal (mean score 7.9; mean EDR96.1) and aneuploid (mean score 8.1; mean EDR, 92.1) embryos.Numbers of hyperploid (n = 17) and hypoploid (n= 11) embryos(non-mosaic cases involving single chromosomes) were not statisticallydifferent. The relative proportions of chromosomes involvedin trisomic karyotypes showed a remarkable similarity to thepattern in spontaneous abortions. Pronuclear status was an unreliablepredictor of ploidy. Small numbers of karyotyped triploid embryosrevealed equal proportions of XXX, XXY and XYY embryos     

Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH

The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1-2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities detected on Day 3. A total of 37 frozen-thawed embryos were analysed in this study. One gave no CGH or FISH results and was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.     

Anaphase lagging mainly explains chromosomal mosaicism in human preimplantation embryos

BACKGROUND: Cleavage stage embryos as well as postimplantation embryos have been studied extensively over the years. However, our knowledge with respect to the chromosomal constitution of human embryos at the blastocyst stage is still rudimentary. METHODS: In the present paper, a large series of human blastocysts was examined by means of fluorescent in situ hybridization (FISH). RESULTS: It was found that only one in four blastocysts (25%) displayed a normal chromosomal pattern. We defined a group of blastocysts (26%) displaying a simple mosaic chromosome pattern (different cell lines resulting from one chromosomal error), an about equally large group of blastocysts (31%) displaying a complex mosaic chromosome pattern, and a smaller group of blastocysts (11%) showing a chaotic chromosome distribution pattern. Six per cent of all blastocysts analysed could not be assigned one of the previously mentioned chromosomal patterns. CONCLUSION: Anaphase lagging appeared to be the major mechanism through which human embryos acquire a mosaic chromosome pattern during preimplantation development to the blastocyst stage.     

Detailed FISH analysis of day 5 human embryos reveals the mechanisms leading to mosaic aneuploidy

BACKGROUND: Fluorescence in situ hybridization (FISH) analysis has shown that human embryos display a high level of chromosomal mosaicism at all preimplantation stages. The aim of this study was to investigate the mechanisms involved by the use of two probes for each of three autosomes at different loci and to determine the true level of aneuploid mosaicism by excluding FISH artefacts. METHODS: Embryos were cultured in two different types of medium: group I were cultured in standard cleavage medium for up to day 5 and group II were cultured from day 3 to day 5 in blastocyst medium. Three rounds of FISH were performed. In round 1, the probes used were 1pTel, 11qTel and 18CEP; in round 2, the probes used were 1satII/III, 11CEP and 18qTel; in round 3, the probes used were 18CEP, XCEP and YCEP. RESULTS: A total of 21 embryos were analysed in each group. The FISH results revealed one uniformly diploid and 20 mosaic embryos for group I, and two uniformly diploid and 19 mosaic embryos for group II. The predominant type of mosaicism was diploid/aneuploid. The use of two different probes per autosome was able to distinguish FISH artefacts affecting 5% of nuclei from true single cell anomalies. CONCLUSIONS: Post-zygotic chromosome loss was the most common mechanism leading to aneuploidy mosaicism for both groups, followed by chromosome gain, with fewer examples of mitotic non-disjunction.     

Chromosome abnormalities in human embryos

The presence of numerical chromosome abnormalities in human embryos was studied using fluorescence in-situ hybridization with four or more chromosome-specific probes. When most cells of an embryo are analysed, this technique allows differentiation to be made between aneuploidy, mosaicism, haploidy and polyploidy. Abnormal types of fertilization, such as unipronucleated, tripronucleated zygotes and zygotes with uneven pronuclei, were studied using this technique. We have found a strong correlation between some types of dysmorphism with chromosomal abnormalities. In addition, the more impaired the development of an embryo, the more chromosomal abnormalities were detected in those embryos. Maternal age and other factors were linked to an increase in chromosome abnormalities (hormonal regimes, temperature changes), but not to intracytoplasmic sperm injection.     

Fertilization and early embryology: Diagnosis of major chromosome aneuploidies in human preimplantation embryos

A short fluorescence in-situ hybridization (FISH) procedureusing fluorochrome and digoxigenin labelled DNA probes was developedfor application in human preimplantation embryos in order toanalyse the five chromosomes most involved in human aneuploidy(X, Y, 18, 13 and 21). The chromosomes were fluorescent-stainedand detected simultaneously in 157 blastomeres from 30 humanembryos. Successful FISH analysis was achieved in 93% of theblastomeres. Aberrations for these chromosomes were found in70% of abnormally developing monospermic embryos. The majorityof normally developing monospermic embryos obtained from olderpatients were also chromosomally abnormal. By analysing allor most of the cells from these embryos, true mosaicism wasdistinguished from technique failure. Mosaic embryos, polyploidembryos with ploidies as high as 8n, haploid embryos, embryosmonosomic for 13/21 and for X, and embryos trisomic for 13/21and 18, were common in abnormally developing embryos. In contrast,aneuploidy was the main chromosome abnormality found in normallydeveloping monospermic embryos.     

The frequency of chromosome anomalies in human preimplantation embryos after in-vitro fertilization

Previous studies have reported chromosome aberrations in humanpre-embryos after in-vitro fertilization (IVF). Although thereason for these abnormalities is not clear, there is evidencethat they can arise during gametogenesis, fertilization or cleavage.The present study has examined further the incidence of chromosomeabnormalities in human pre-embryos after IVF, using oocytesrecovered from normal volunteer women and from women undergoinginfertility treatment in an embryo-replacement programme. Chromosomepreparations were performed for 75 pre-embryos. Of these 35(47%) gave at least one metaphase in which analysis was possible.The overall incidence of abnormal pre-embryos was 40% (14/35).The absolute frequency of aberrations was 9% for trisomies,3% for polyploidies, 26% for structural anomalies and 3% forhypodiploidies. Five pre-embryos were found to be mosaics, threeof which had each one trisomic metaphase. In five of the pre-embryosmultiple anomalies were found. In 13 of the 14 abnormal pre-embryosthe aberrations were found in only one metaphase. The presentstudy demonstrates that trisomic mosaicism may not be a rareevent in human pre-embryos. Further evidence is provided thatmitotic non-disjunction is important for the production of aberrationsin human pre-embryos     

CB-FISH法检测人体内嵌合的21号染色体非整倍体

目的　探讨 2 1三体患者体内二倍体细胞增多的机理。方法　用松胞素 - B诱导的双核细胞荧光原位杂交技术 ,对人体内 2 1号染色体非整倍体和培养细胞中 2 1号染色体分离异常的发生率进行检测。结果　 2 1三体患者体内 ,二倍体细胞率 (16 .90‰± 10 .70‰ )远高于 2 1四体细胞率 (0 .42‰± 0 .6 4‰ ) (P=0 .0 0 0 )。在培养的正常人和 2 1三体患者细胞中 ,2 1号染色体不分离率 (3.6 9‰± 2 .5 0‰和 8.2 2‰±5 .5 4‰ )显著高于丢失率 (0 .10‰± 0 .30‰和 0 .43‰± 0 .49‰ ) (P=0 .0 0 0 )。结论　 2 1三体患者体内较高比例的二倍体细胞源于因三体细胞中 2 1号染色体分离异常而形成的二倍体细胞的逐渐积累     

Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells

Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.     

Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro

BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro. METHODS: Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS: These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.     

Chromosome analysis of human oocytes and embryos: does delayed fertilization increase chromosome imbalance?

Thirty per cent of a sample of 120 unfertilized human oocytescarried chromosome abnormalities highly correlated with maternalage (38% in patients >35, as compared with 24% in youngerpatients). Fertilized eggs, when observed 17 h after insemination,showed in 1.6% a single pronudeus suggesting parthenogeitelicactivation. In 92% of the cases two pronuclei were observedand the rate of chromosome anomalies depended on the morphologicalaspect of the embryos. Triploidy was also encountered in 6.4%of the eggs leading to an overall rate of chromosome aberrationsreaching 29.2%. Delayed fertilization drastically increasedthe rate of chromosome anomalIes (87%) as well as the rate ofmosaicism: 30% versus 10.6% in timely fertilized eggs. The highrate of chromosome disorders in early life after in-vitro fertilization(IVF) raises the ethical question of the opportunity of carryingout a genetic control of normality in human embryos at the preimplantationstage.     

Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization

We have evaluated the incidence of chromosome 21 disomy in decondensedsperm heads from nine normal men using a locus-specific DNAprobe for chromosome 21, and a centromeric probe for chromosome6 as a control. The results show that the incidence of chromosome21 disomy (0.38%) is significantly higher than disomy for chromosome6 (0.14%). No differences were found among the individuals analysed.     

Post-fertilization polyploidy in human preimplantation embryos fertilized in-vitro

The development of in-vitro fertilization (IVF) and embryo transfer(ET) as a treatment for certain forms of infertility has emphasizedthe need for more information on why so many preimplantationembryos fail to develop beyond the very early cleavage stages.Analysis of nuclei from embryos that had ceased to grow or thatwere morphologically abnormal was carried out using one of twotechniques: measurement of the DNA content of their nuclei,or observation of the number of Y chromasomes per nucleus. Itwas found that there was a correlation between the length oftime in culture and abnormal development of polyploidy in anumber of the cells. On the other hand, a small proportion ofpolyploid cells observed in a morphologically normal expandedblastocyst probably represents the normal precursors of polyploidtrophoblast cells as observed in other mammalian species.     

Rapid in situ detection of chromosome 21 by PRINS technique

The “PRimed IN Situ labeling” (PRINS) method is an interesting alternative to in situ hybridization for chromosomal detection. In this procedure, chromosome labeling is performed by in situ annealing of specific oligonucleotide primers, followed by primer elongation by a Taq polymerase in the presence of labeled nucleotides. Using this process, we have developed a simple and semi-automatic method for rapid in situ detection of human chromosome 21. The reaction was performed on a programmable temperature cycler, with a chromosome 21 specific oligonucleotide primer. Different samples of normal and trisomic lymphocytes and amniotic fluid cells were used for testing the method. Specific labeling of chromosome 21 was obtained in both metaphases and inter-phase nuclei in a 1 hour reaction. The use of oligonucleotide primer for in situ labeling overcomes the need for complex preparations of specific DNA probes. The present results demonstrate that PRINS may be a simple and reliable technique for rapidly detecting aneuploidies. © 1995 Wiley-Liss, Inc.     

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.     

Mitotic and meiotic behaviour of a naturally transmitted ring Y chromosome: reproductive risk evaluation

BACKGROUND: The mitotic and meiotic behaviour of a transmitted ring Y [r(Y)] chromosome from a father to his Klinefelter syndrome (KS) son, and the mechanism of ring formation are analysed herein. To our knowledge, this is the first reported case of natural transmission of an r(Y). METHODS AND RESULTS: Amplification of X chromosome polymorphisms by PCR showed that the KS was of paternal origin. G-banding and fluorescence in situ hybridization (FISH) studies revealed a similar percentage of mosaicism in father and son by mitotic loss of r(Y). SRY gene and Y marker amplification by PCR, FISH with subtelomeric probes for Xp/Yp and Xq/Yq, and comparative genomic hybridization (CGH) analyses indicated the intactness of the Y chromosome from SRY to subtelomere Yq. FISH analysis of sperm from the father showed significantly higher frequencies (P<0.005) for diploidy and for 6, 13, 18, 21, 22, XX, XY disomies than those observed in control donors. CONCLUSIONS: An r(Y) with low material loss can be naturally transmitted, showing similar mitotic behaviour in the offspring. The presence of an r(Y) chromosome in germinal cells increased the risk of fathering offspring with numerical abnormalities, even for chromosomes not involved in the arrangement.