Chromosome constitution of human embryos generated after in vitro maturation including 3-isobutyl-1-methylxanthine in the oocyte collection medium.

STUDY QUESTION: Do cleavage-stage embryos obtained from oocytes matured in vitro after pre-incubation with a phosphodiesterase inhibitor (IBMX) carry more chromosomal abnormalities than those generated from oocytes matured in vivo? SUMMARY ANSWER: The rate and type of chromosomal abnormalities in normally developing cleavage-stage embryos generated with an in vitro maturation (IVM) system including pre-incubation with IBMX are not different from those observed in supernumerary embryos obtained from oocytes matured in vivo. WHAT IS KNOWN ALREADY: Very limited information is available about the chromosomal constitution of IVM embryos. Previous studies were carried out using FISH on single biopsied blastomeres or arrested whole embryos and only provided fragmentary information on chromosomal abnormalities in IVM embryos. There is no systematic study of chromosomal abnormalities in all blastomeres of human Day 3 embryos with good morphology. STUDY DESIGN, SIZE, DURATION: Between July 2012 and December 2012, 16 young (age <35 years old) egg donors underwent 18 IVM cycles for the generation of research embryos. Eighteen embryos developed to Day 3 and were analysed using array comparative genomic hybridization (aCGH). PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature oocytes were retrieved from 2 to 10 mm follicles after mild ovarian stimulation with gonadotrophins but without hCG ovulation trigger. At collection, oocytes were pre-incubated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and matured in vitro. After IVM culture, mature oocytes were microinjected with sperm from a single donor. Embryos were cultured to Day 3 after ICSI and all blastomeres of 18 good-morphology embryos were collected individually for aCGH. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte maturation rate in vitro was 50.2% (120/239). The mean fertilization rate was 68.3% (82/120) and 30.5% (25/82) of fertilized oocytes developed into a morphologically good quality embryo on Day 3 after ICSI. Of these, 18 embryos that developed well up to Day 3 were analysed using aCGH. Eighty of the 123 blastomeres analysed showed at least one chromosomal abnormality. Three out of eighteen embryos had completely normal cells. A single embryo carried a meiotic abnormality, 11 embryos were mosaic and three were chaotic. Although the aneuploidy data of this study are too limited to allow statistical analysis, these data are comparable to our own published data on the chromosome constitution of whole day 3 and day 4 embryos after conventional ART. LIMITATIONS, REASONS FOR CAUTION: Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. Although the limited number of embryos analysed here precludes firm conclusions, they provide valuable data on possible causes of the reduced potential of IVM embryos. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to describe the complete chromosome complement of all single blastomeres of good-morphology day 3 embryos obtained with IVM (including the presence of IBMX in a pre-incubation medium). The results demonstrate that a high proportion of good-morphology embryos are aneuploid and that there is no obvious increase in aneuploidies as a result of IVM which seems to suggest that the reduced efficiency of IVM technology compared with standard IVF may be accounted for by factors other than aneuploidy, such as cytoplasmic defects or reduced endometrial receptivity. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the TBM (Applied Biomedical Research with Societal Finality) programme of the IWT (Agency for Innovation through Science and Technology - Flanders, 110680) and by a Methusalem grant of the Vrije Universiteit Brussel. C.S. is a post-doctoral fellow of the Fund for Scientific Research Flanders (FWO - Vlaanderen). K.J. is a PhD student funded by the FWO. The University of Adelaide owns a patent family associated with IVM technologies that is licensed to Cook Medical. R.B.G. and J.G.T. are inventors. The remaining authors have no conflict of interest to declare.

Gain of 20q11.21 in human embryonic stem cells improves cell survival by increased expression of Bcl-xL.

Gain of 20q11.21 is a chromosomal abnormality that is recurrently found in human pluripotent stem cells and cancers, strongly suggesting that this mutation confers a proliferative or survival advantage to these cells. In this work we studied three human embryonic stem cell (hESC) lines that acquired a gain of 20q11.21 during in vitro culture. The study of the mRNA gene expression levels of the loci located in the common region of duplication showed that HM13, ID1, BCL2L1, KIF3B and the immature form of the micro-RNA miR-1825 were up-regulated in mutant cells. ID1 and BCL2L1 were further studied as potential drivers of the phenotype of hESC with a 20q11.21 gain. We found no increase in the protein levels of ID1, nor the downstream effects expected from over-expression of this gene. On the other hand, hESC with a gain of 20q11.21 had on average a 3-fold increase of Bcl-xL (the anti-apoptotic isoform of BCL2L1) protein levels. The mutant hESC underwent 2- to 3-fold less apoptosis upon loss of cell-to-cell contact and were ∼2-fold more efficient in forming colonies from a single cell. The key role of BCL2L1 in this mutation was further confirmed by transgenic over-expression of BCL2L1 in the wild-type cells, leading to apoptosis-resistant cells, and BCL2L1-knock-down in the mutant hESC, resulting in a restoration of the wild-type phenotype. This resistance to apoptosis supposes a significant advantage for the mutant cells, explaining the high frequency of gains of 20q11.21 in human pluripotent stem cells.

Evolution of aneuploidy up to Day 4 of human preimplantation development.

STUDY QUESTION: What is the incidence of aneuploidy and mosaicism in all cells of top-quality Day-4 embryos analysed by array-based comparative genomic hybridization (array CGH)? SUMMARY ANSWER: Our data show extensive abnormalities in Day-4 embryos. WHAT IS KNOWN ALREADY: Numerous studies on human embryos at Day 3 and Day 5 of development show that they frequently contain aneuploid cells and are mosaic, although Day-5 embryos contain proportionally more normal cells than at Day 3. In contrast, only limited data exist on Day 4 of preimplantation development, despite the fact that it is the suggested stage for the initiation of the process of self-correction. STUDY DESIGN, SIZE, DURATION: Thirteen embryos were analysed: four fresh good-quality preimplantation genetic diagnosis (PGD) embryos and nine good-quality surplus embryos cryopreserved on Day 3 and donated for research. On Day 4, following removal of the zona pellucida, all blastomeres were disaggregated and collected. PARTICIPANTS/MATERIALS, SETTING, METHODS: The genomic DNA of 283 single blastomeres from disaggregated embryos was amplified. Array CGH was carried out using 24SureTM Cytochip microarrays. After scanning of the microarray slides, the images were analysed using BlueFuse Software (BlueGnome). Combined with selective microsatellite analysis, hypothetical reconstructions of embryo chromosome complements were made following each of the first four cleavage divisions. MAIN RESULTS AND THE ROLE OF CHANCE: No chromosome imbalance was detected for one PGD embryo, the other three were mosaic containing between 16 and 75% abnormal cells. All nine frozen-thawed embryos were abnormal. Six were mosaic with between 30 and 100% abnormal cells; three had abnormalities of meiotic origin, two of which displayed mitotic abnormalities. Evidence was also found of mitotic unbalanced structural chromosome rearrangements. The higher rate of abnormality of frozen-thawed embryos is based on a small number of embryos and cannot be tested statistically. The aneuploidy can mostly be explained by anaphase lag and non-disjunction. In some cases, we hypothesize endoreduplication followed by a cellular division with multipolar spindles to explain the results. LIMITATIONS, REASONS FOR CAUTION: Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. The division trees presented are hypothetical models projecting back in time that try to explain observations in single blastomeres of Day 4 embryos. The limited number of embryos analysed does not allow drawing firm conclusions, but nevertheless provides valuable data on the origin of aneuploidy in human embryos. WIDER IMPLICATIONS OF THE FINDINGS: Our data show extensive abnormalities in Day-4 embryos. We found no evidence of self-correction at this stage of development, suggesting that this process may start at a later stage of development.

Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos.

STUDY QUESTION: What are the aneuploidy rates and incidence of mosaicism in good-quality human preimplantation embryos. SUMMARY ANSWER: High-level mosaicism and structural aberrations are not restricted to arrested or poorly developing embryos but are also common in good-quality IVF embryos. WHAT IS KNOWN ALREADY: Humans, compared with other mammals, have a poor fertility rate, and even IVF treatments have a relatively low success rate. It is known that human gametes and early preimplantation embryos carry chromosomal abnormalities that are thought to lower their developmental potential. STUDY DESIGN, SIZE AND DURATION: The embryos studied came from nine young (age <35 years old) IVF patients and were part of a cohort of embryos that all resulted in healthy births. These 14 embryos inseminated by ICSI and cryopreserved on Day 2 of development were thawed, cultured overnight and allowed to succumb by being left at room temperature for 24 h. Following removal of the zona pellucida, blastomeres were disaggregated and collected. PARTICIPANTS/MATERIALS, SETTING AND METHODS: There were 91 single blastomeres collected and amplified by multiple displacement amplification. Array-comparative genomic hybridization was performed on the amplified DNA. Array-data were normalized and aneuploidy was detected by the circular binary segmentation method. MAIN RESULTS AND THE ROLE OF CHANCE: The good-quality embryos exhibited high rates of aneuploidy, 10 of 14 (71.4%) of the embryos being mosaic. While none of the embryos had the same aneuploidy pattern in all cells, 4 of 14 (28.6%) were uniformly diploid. Of the 70 analysed blastomeres, 55.7% were diploid and 44.3% had chromosomal abnormalities, while 29% of the abnormal cells carried structural aberrations. WIDER IMPLICATIONS OF THE FINDINGS: Finding such a high rate of aneuploidy and mosaicism in excellent quality embryos from cycles with a high implantation rate warrants further research on the origin and significance of chromosomal abnormalities in human preimplantation embryos. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Instituut voor de aanmoediging van innovatie door Wetenschap en Technologie in Vlaanderen (IWT-Vlaanderen). A.M. is a PhD student at the IWT-Vlaanderen. C.S. is a postdoctoral fellow at the FWO Vlaanderen. There are no competing interests.

Characterization of CD30 expression in human embryonic stem cell lines cultured in serum-free media and passaged mechanically.

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30, reported to be a biomarker of hESCs with abnormal karyotype, in undifferentiated and spontaneously differentiated hESC. METHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC, even at very early passages, without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC. CONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions, and that KO-SR may play a role. In addition, the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities.