Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.     

Integrins and adhesion molecules: Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos

Human embryos have been biopsied at either the cleavage or theblastocyst stage of development. One to two blastomeres wereremoved from cleavage-stage embryos and 2–6 cells fromblastocysts. The biopsy specimens were subjected to gene amplificationby the polymerase chain reaction (PCR) and a comparison madeof amplification efficiencies of two unique target sequences,one located within the -globin gene and containing the sickle-celllocus and the other a polymorphic dinucleotide repeat. Whenthe cleavage-stage biopsy sample consisted of an intact blastomerewith a clearly discernible nucleus, an amplification efficiencyof 89% was achieved for each target locus. This was similarto that achieved with cleavage-stage biopsy samples consistingof two blastomeres or with blastocyst biopsy samples consistingof 2–3 trophectoderm cells. When biopsy samples consistedof four or more trophectoderm cells, both target loci were amplifiedin all samples tested. When the biopsy sample was heterozygousat the dinucleotide repeat locus and the biopsy consisted ofone or more intact cells with a clearly discernible nucleus,both alleles were amplified in >80% of biopsy samples. Whenfour or more trophectoderm cells were used for the PCR, bothalleles were amplified in all heterozygous samples. Target sequenceswere never amplified from biopsy samples which lysed prior totransfer into the reaction tube. Analysis of DNA fragments amplifiedfrom the dinucleotide repeat locus indicated that in most casesfaithful amplification of biopsy DNA template had taken place.However, in one case, fragments were identified which couldnot have resulted from the amplification of embryonic sequencesalone, indicating that contamination with extraneous DNA mayhave taken place. The significance of this finding for therapeuticpreimplantation genetic diagnosis is discussed.     

Preimplantation diagnosis of a human beta-globin transgene in biopsied trophectoderm cells and blastomeres of the mouse embryo.

The preimplantation diagnosis of a HbSA-globin transgene in biopsied trophectoderm cells and blastomeres in embryos using a transgenic mouse model for the trait of human sickle-cell anaemia has been undertaken. A sensitive procedure was developed for the amplification of the human beta-globin gene sequence flanking the sickle mutation. Polymerase chain reaction (PCR) assays were undertaken on one to five biopsied trophectoderm cells and isolated blastomeres of the preimplantation mouse embryo. After biopsy the blastocysts were cultured whilst the cells were analysed for the presence of the transgene, and a high proportion (82-91%) were viable as assessed by the presence of a blastocoele cavity within a 5-h period. The majority of the biopsied cultured blastocysts were frozen and used to confirm the diagnosis; 90 biopsied cultured blastocysts were transferred to pseudopregnant recipients and 34% established pregnancy. Material from day 13.5 post-coitum fetuses was also used to confirm the original diagnosis. The time (4-5 h) required to carry out the analysis obviates a need for extended culture or cryopreservation of the biopsied embryo. In individual experiments under optimal conditions, the presence of the transgene in biopsied cells was detected with 100% accuracy, and the PCR analysis was sensitive at the 1-cell level. The overall success rate of diagnosis and confirmation of the presence or absence of the human beta-globin sequence in the biopsied embryo was 70%. Over the entire experimental period (14 months) DNA contamination from a variety of sources did occasionally occur; the methods used to overcome this problem are discussed.     

DNA fingerprinting of sister blastomeres from human IVF embryos

BACKGROUND: Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis. METHODS: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n = 50) and aneuploid (n = 25) buccal cells and embryonic blastomeres (n = 21). RESULTS: The optimized pentaplex PCR DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA (<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down's syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental alleles. CONCLUSIONS: A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic microsatellite markers was developed for single cells. This finding paves the way for the development of a more complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from couples at genetic risk.     

Mutation and haplotype analysis for Duchenne muscular dystrophy by single cell multiple displacement amplification

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder with mutational heterogeneity. The scarcity of DNA from single cells in preimplantation genetic diagnosis (PGD) for DMD limits comprehensive genetic testing. Multiple displacement amplification (MDA) is reported to generate large amounts of template and give the most complete coverage and unbiased amplification to date. Here, we developed mutation and haplotype analysis in conjunction with gender determination on MDA products of single cells providing a generic approach that widens availability of PGD for female carriers with varied mutations. MDA amplified with 98.5% success for single lymphocytes and 94.2% success for single blastomeres, which was evaluated on 60 lymphocytes and 40 blastomeres. A total of six commonly mutant exons, eight short tandem repeat markers within dystrophin gene and amelogenin were incorporated into subsequent singleplex PCR assays. The mean allele dropout rate was 9.0% for single lymphocytes and 25.5% for single blastomeres. None of the blank controls gave a positive signal. Genotyping of each pedigree for three families provided 2-3 fully informative alleles per dystrophin haplotype besides specific mutant exons and amelogenin. We suggest that this approach is reliable to identify non-carrier female embryos other than unaffected male embryos and reduce the risk of misdiagnosis.     

Preimplantation genetic diagnosis of spinocerebellar ataxia 3 by (CAG)(n) repeat detection

Spinocerebellar ataxia 3 (SCA3) is an autosomal dominant neurodegenerative disorder characterized by variable expression and a variable age of onset. SCA3/MJD (Machado-Joseph disease) is caused by an expansion of a (CAG)(n) repeat in the MJD1 gene on chromosome 14q32.1. A single cell PCR protocol has been developed for preimplantation genetic diagnosis (PGD) of SCA3 to select unaffected embryos on the basis of the CAG genotype. Single leukocytes and blastomeres served as a single cell amplification test system to determine the percentage of allelic drop-out (ADO) and PCR efficiency. Out of 105 tested heterozygous single leukocytes, 103 (98.1%) showed a positive amplification signal, while five cells (4.9%) showed ADO. Amplification in single blastomeres was obtained in 13 out of a total of 14, and ADO was observed in two out of the 13 single blastomeres. PGD of SCA3 was performed in a couple with paternal transmission of the SCA3 allele. Seven embryos were available for biopsy, all biopsied blastomeres showed amplification and no ADO occurred. One embryo was diagnosed as affected whereas six embryos were diagnosed as unaffected. Two unaffected embryos were transferred and resulted in a singleton pregnancy and the birth of a healthy girl.     

Reliable gender screening for human preimplantation embryos, using multiple DNA target-sequences.

Dependable methods were developed for preimplantation sexing of human IVF embryos, for use in clinical settings where prospective parents are at high risk for transmission of X-linked diseases. Using single cultured cells and blastomeres from human embryos as model systems, a multiplex protocol was developed for rapid analysis via nested polymerase chain reaction (PCR). Reliability was enhanced by co-amplification of conserved amelogenin gene segments from both X and Y chromosomes, as well as Y-linked DYZ1 repetitive elements. Each cell was manually isolated and individually washed to avoid potential contaminants. Multiplex amplification allowed recognition of spurious amplification failures specific to particular amelogenin single-copy targets. The X-linked internal control and multiple Y-linked markers allowed recognition and exclusion of most aberrant samples, thus averting potential misdiagnosis. The optimized single-cell protocol reduced experimental sexing errors to < 2% (1/60), but also revealed potential pitfalls of single-cell analysis. With human triploid embryos, separate sampling of individual blastomeres provided concordant female or male signals. Slight modification adapted the procedure for diagnosis of biopsy material from blastocyst stage embryos, allowing separate analysis of multiple tubes containing multiple cells for improved reliability.     

Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs

The present study was aimed to facilitate karyotyping of human blastomeres using the metaphase-inducing factors present in unfertilized eggs. A rapid technique for karyotyping would have wide application in the field of preimplantation genetic diagnosis. When cryopreserved in-vitro matured bovine oocytes were fused with human blastomeres, the transferred human nuclei were forced into metaphase within a few hours. Eighty-seven human blastomeres from abnormal or arrested embryos were fused with bovine oocytes in a preclinical study. Fusion efficiency was 100%. In 21 of the hybrid cells, no trace of human chromatin was found. Of the remaining 66, 64 (97%) yielded chromosomes suitable for analysis. The method was used to karyotype embryos from two patients with maternal translocations. One embryo which was judged to be karyotypically normal was replaced in the first patient, resulting in one pregnancy with a normal fetus. None of the second patient's embryos was diagnosed as normal, and hence none was transferred. The results of the present study demonstrated that the ooplasmic factors which induce and maintain metaphase in bovine oocytes can force transferred human blastomere nuclei into premature metaphase, providing the basis for a rapid method of karyotyping blastomeres from preimplantation embryos and, by implication, cells from other sources.     

Caspase activity in preimplantation human embryos is not associated with apoptosis

BACKGROUND: Previous studies on mammalian preimplantation embryos have suggested an association between caspase activation, blastomere fragmentation and apoptosis. However, some reports on human embryos questioned the causal relationship between blastomere fragmentation and apoptosis, and information about the presence and activity of caspases in human embryos is lacking. METHODS: A fluorochrome-labelled universal caspase inhibitor was used to visualize active caspases in blastomeres and fragments of preimplantation human embryos. RESULTS: Caspase activity was detected only after fertilization, and was rare in blastomeres but frequent in fragments. The incidence of caspase activity in blastomeres and fragments was stable between the 2-cell and 12-cell stages. Caspase-positive blastomeres were only seen in poor-morphology embryos. The percentage of caspase-positive fragments was increased in embryos with multinucleated blastomeres but was unrelated to embryo morphology. Moreover, caspase-positive fragments detached from healthy blastomeres that were isolated by embryo biopsy and subsequently underwent mitotic division in culture. CONCLUSIONS: These data suggest that caspases in preimplantation human embryos are involved in developmental processes unrelated to cell death.     

Use of a polymorphic dinucleotide repeat sequence to detect non-blastomeric contamination of the polymerase chain reaction in biopsy samples for preimplantation diagnosis

Using the polymerase chain reaction (PCR), amplification oftwo different target DNA sequences has been achieved with highfrequency using single human blastomeres as template for theduplex reaction. One sequence is located within the –globingene and contains the sickle cell locus, the other is a polymorphicdinucleotide repeat, which, as well as acting as a positivecontrol for amplification, was used to check the origin of theamplified DNA. A comparison of the sequences amplified fromthe blastomere with sequences amplified from parental samplesconfirmed that amplification of blastomeric sequences, but notextraneous contaminating DNA, had taken place in most cases.The efficacy of this system for detecting extraneous DNA waschecked by deliberately contaminating single blastomeres withforeign cells. The presence of contamination was detected bythe amplification of sequences not present in blastomeric DNAand which therefore must have been amplified from extraneouscontaminating DNA.     

Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major

PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for beta-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.     

Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study

BACKGROUND: Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for beta-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts. METHODS: This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis. RESULTS: In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born. CONCLUSIONS: This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.     

Traditional detection versus computer-controlled multilevel analysis of nuclear structures from donated human embryos

BACKGROUND: Multinuclearity is known to correlate with decreased implantation and pregnancy rates. Thus, a valid detection of nuclear structures especially among otherwise good quality embryos may be of great importance in order to improve clinical outcome. In this study, we have compared traditional manual microscopic analysis with computer-controlled multilevel morphological assessment for analysis of nuclear status in human embryos. METHODS: In total, 84 donated 2- and 4-cell embryos with < or = 20% fragmentation from patients referred for IVF or ICSI treatment were included. Mono- and multinuclearity was recorded using traditional analysis as well as computer-controlled multilevel analysis of each intact embryo. Subsequently, the embryos were separated into individual blastomeres to assess the number of nuclear structures. All nuclear structures were fixed and stained for DNA. RESULTS: There was no significant difference (P = 1.0) between embryonic nuclear status detected by computer-controlled analysis of the intact embryos and of the separated blastomeres. Additionally, 100% of the fixed nuclear structures contained DNA. However, using traditional morphological analysis, significantly more embryos (26%) had incorrect nuclear status detected (P = 0.002). Further, the presence of <10% embryonic fragmentation had no impact on the correct detection of nuclear structures using the multilevel analysis. For embryos with 11-20% fragmentation, 86% of the nuclear structures detected in the separated blastomeres were found in the intact embryos. The mean diameter of nuclear structures was significantly decreased from 22.1 microm in mononucleate 2-cell embryos to 18.7 microm in mononucleate 4-cell embryos (P < 0.001). CONCLUSION: The results of this study indicate that the use of computer-controlled multilevel morphological analysis can improve the detection of nuclear structures in human embryos.     

Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos

Human embryos have been biopsied at either the cleavage or theblastocyst stage of development. One to two blastomeres wereremoved from cleavage-stage embryos and 2–6 cells fromblastocysts. The biopsy specimens were subjected to gene amplificationby the polymerase chain reaction (PCR) and a comparison madeof amplification efficiencies of two unique target sequences,one located within the ß-globin gene and containingthe sickle-cell locus and the other a polymorphic dinucleotiderepeat. When the cleavage-stage biopsy sample consisted of anintact blastomere with a clearly discernible nucleus, an amplificationefficiency of 89% was achieved for each target locus. This wassimilar to that achieved with cleavage-stage biopsy samplesconsisting of two blastomeres or with blastocyst biopsy samplesconsisting of 2–3 trophectoderm cells. When biopsy samplesconsisted of four or more trophectoderm cells, both target lociwere amplified in all samples tested. When the biopsy samplewas heterozygous at the dinucleotide repeat locus and the biopsyconsisted of one or more intact cells with a clearly discerniblenucleus, both alleles were amplified in >80% of biopsy samples.When four or more trophectoderm cells were used for the PCR,both alleles were amplified in all heterozygous samples. Targetsequences were never amplified from biopsy samples which lysedprior to transfer into the reaction tube. Analysis of DNA fragmentsamplified from the dinucleotide repeat locus indicated thatin most cases faithful amplification of biopsy DNA templatehad taken place. However, in one case, fragments were identifiedwhich could not have resulted from the amplification of embryonicsequences alone, indicating that contamination with extraneousDNA may have taken place. The significance of this finding fortherapeutic preimplantation genetic diagnosis is discussed. ß-globin/CA repeat/embryo biopsy/polymerase chain reaction     

Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes

Dual fluorescent in-situ hybridization (FISH) using X and Ychromosome specific probes has been used to identify the sexof human embryos for preimplantation diagnosis of X-linked disease.With a modified spreading method and directly labelled fluorescentDNA probes, we have examined the possibility of reducing thetime of the FISH procedure from 7 to 2 h. A total of 17 normallyfertilized human embryos were disaggregated and 98 intact blastomeresobtained. The spreading efficiency was 96% and FISH signalswere obtained from 97% of nuclei. In all cases, sibling blastomeresfrom the same embryo were the same sex. Mosaicism was observedin some embryos. Five cells which lysed during the dis–aggregationprocess were spread to determine whether FISH was possible inthese cells, but in all cases the morphology of the nuclei waspoor and multiple signals were observed so that reliable diagnosisof sex was not possible. The data reported here confirm thatby using an improved spreading method in combination with directlylabelled DNA probes, we have increased the efficiency and reducedthe time required for sexing embryos for preimplantation diagnosisof X-linked disease.     

Preimplantation development of mouse and human embryos biopsied at cleavage stages using a modified displacement technique

A modified embryo biopsy method was tested on four- and eight-cell stage mouse embryos and used on human embryos to obtain blastomeres for preimplantation genetic diagnosis. The biopsy method tested combines zona drilling and fluid displacement to force one or two cells through an opening in the zona pellucida of the cleavage-stage embryo. Rates of cell division and the percentage of mouse embryos forming blastocysts following biopsy at the eight-cell stage were not significantly different from those observed in unoperated control embryos. The percentage blastocyst formation was not significantly different in embryos biopsied at the four-cell stage and in control embryos, although cell division was significantly retarded following biopsy. 96% of the mouse blastomeres isolated at the eight-cell stage were recovered intact and 96% of those placed in culture underwent cell division. Survival and division of cells isolated at the four-cell stage were 92 and 84% respectively. Most of the cultured blastomeres cleaved several times and formed small trophoblast vesicles. Chromosomes were observed in 59% of blastomeres incubated in the presence of colcemid. In the initial use of this biopsy technique for human preimplantation genetic diagnosis, blastocyst formation was observed in 9 of 13 human embryos biopsied at the 7- to 10-cell stage. These findings support the use of this biopsy method as an alternative to aspiration techniques.      

Genetic diagnosis using polymerase chain reaction and fluorescent in-situ hybridization analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos

Cultured human preimplantation embryos have been used to developmethods which allow preimplantation genetic diagnosis (PGD)analyses by polymerase chain reaction (PCR) and fluorescentin-situ hybridization (FISH) on biopsied blastomeres and trophectodermcells from the same embryo. An experimental design is describedand experiments undertaken, which demonstrate the feasibilityof extending biopsy and PGD procedures currently in use. Wehave shown that dual-stage biopsies are possible, and that thePCR and FISH analyses of the biopsied cell samples are effective.One to two blastomeres were biopsied from an 8- to 10-cell embryoand processed for the simultaneous PCR amplification of a -globinand a cytosine adenine (CA) repeat sequence, or a Y chromosomesequence. FISH procedures were also used to detect the presenceof Y chromosome markers. The biopsied cleavage-stage embryocan be cultured to the blastocyst stage, where the serial biopsyof three to five mural trophectoderm cells provides two furthercell samples. These can be used to repeat and/or undertake additionalPGD analyses. The biopsied blastocyst is either used to confirmearlier diagnoses, or placed in culture for a further 4–24h. Maintenance of a blastocoele cavity, hatching and formationof an outgrowth demonstrates continuing viability followingthe dual-stage biopsy procedures. The PCR DNA amplificationprocedures are effective at the cellular level for both biopsiedblastomeres and mural trophectoderm cells. The FISH techniqueshave shown a definitive Y signal in 50% (one out of two) and100% (two out of two) of the biopsied blastomeres and 72% (twoout of three, four out of five and 7 out of 10) for the trophectodermcell nuclei. Preliminary experiments have demonstrated thatthe FISH preparations can be re-amplified to improve the signal,and dual fluorescent procedures using the X and Y probes areeffective. A retrospective PCR analysis has also been undertakenon preparations of biopsied cells which were previously usedfor PGD analysis by FISH.     

Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells.

We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single lymphocytes and blastomeres from embryos, in order to analyse five exons commonly deleted in deletion-type Duchenne muscular dystrophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex PCR reactions together with the sex-determining region Y (SRY) gene which enables simultaneous gender assignment. We present here PCR amplification results from single lymphocytes isolated from a normal male (220 cells), a normal female (24 cells) and a male DMD patient (40 cells) carrying a deletion of exons 46-49 within the DMD gene. The method failed to produce a PCR signal for the SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 0-4.5% of normal male cells. One negative control out of 112 was positive. When this method was used to analyse two blastomeres from each of five embryos, concordant results were obtained for each pair of blastomeres. All embryos produced signals for the DMD exon tested with four of the embryos found to be male and one female. This method is therefore suitable for preimplantation genetic diagnosis and will allow the transfer of healthy embryos (both male and female) in families carrying DMD gene deletions involving at least one of the five exons 17, 19, 44, 45 and 48.     

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.     

Diagnosing and preventing inherited disease: An analysis of multinucleated blastomere formation in human embryos

Human embryos were disaggregated into component blastomeres42–72 h after insemination. The blastomeres were scoredfor the number of nuclei present and blastomeres of known nuclearmorphology were returned to individual culture drops for 16–20h, after which they were scored for cleavage and nuclear morphology.In all, 48% of mononucleated blastomeres cleaved during thisperiod, but only 76% of these produced two mononucleated daughterblastomeres; in the remainder, one or more of the blastomereswas abnormally nucleated. During overnight culture, 30% of multinucleatedblastomeres and 30% of anucleate blastomeres cleaved, the majorityproducing abnormally nucleated daughter blastomeres. The majorityof blastomeres which showed no sign of cleavage after overnightculture retained the same nuclear morphology as when originallydisaggregated. However, a small number of mononucleated blastomerescontained two nuclei after culture, indicating that karyokinesismay have taken place in the absence of cytokinesis. Overall,30% of blastomeres with more than one nucleus seemed to ariseby this mechanism, the remainder probably arising by errorsof chromosome segregation and/or packaging at mitosis. In addition,25/111 mononucleated daughter cells arose either after abnormaldivision of mononucleated parent cells or after division ofmultinucleated cells, suggesting that 23% of newly formed mononucleatedcells might be chromosomally abnormal. The results of DNA quantitationindicated that very few (12/131, 9.2%) blastomeres (whetherunior multinucleated) had a DNA content outside the 2–4Crange. The embryos used for these studies had been culturedin one of three commonly used in-vitro fertilization (IVF) media:modified T6, Earle‘s balanced salts or Universal IVF medium(a commercial medium from Medi-Cult). A retrospective analysiswas carried out of the number of embryos containing multinucleatedblastomeres at disaggregation and of the total proportion ofisolated blastomeres which were multinucleated in three groupsof embryos, each of which had been cultured in one of the IVFmedia. Both these parameters were found to vary between cohortsof embryos cultured in the different media. The mechanism(s)by which culture medium composition might affect multinucleationof human blastomeres is discussed, as is the significance ofthese data for reliable preimplantation diagnosis of geneticstatus.