scholarly journals Tripolar mitosis drives the association between maternal genotypes of PLK4 and aneuploidy in human preimplantation embryos

2017 ◽  
Author(s):  
Rajiv C. McCoy ◽  
Louise J. Newnham ◽  
Christian S. Ottolini ◽  
Eva R. Hoffmann ◽  
Katerina Chatzimeletiou ◽  
...  
Keyword(s):  
Time Lapse ◽  
Preimplantation Embryos ◽  
Chromosomal Mosaicism ◽  
Cleavage Stage ◽  
Preimplantation Genetic Testing ◽  
Centriole Duplication ◽  
Mechanisms Of Formation ◽  
Diploid Cells ◽  
Human Preimplantation Embryos

AbstractAneuploidy is prevalent in human preimplantation embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing in frequency with maternal age. Superimposed on these meiotic aneuploidies are a range of errors occurring during early mitotic divisions of the embryo, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24,653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17,051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking to quantify their incidences and gain insight into their mechanisms of formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of day-3 blastomeres possessing karyotypes suggestive of tripolar mitosis in normally-fertilized diploid zygotes or descendant diploid cells. We further supported this hypothesis using time-lapse data from an intersecting set of 77 cleavage-stage embryos. The diploid tripolar signature was rare among day-5 blastocyst biopsies (0.5%), suggesting that complex aneuploidy generated by tripolar mitosis impairs cellular and/or early embryonic survival. Strikingly, we found that the tripolar mitosis mechanism is responsible for the previously described association with common maternal genetic variants spanning PLK4. Our findings are consistent with the role of PLK4 as the master regulator of centriole duplication with a known capacity to induce tripolar mitosis when mutated or mis-expressed. Taken together, we propose that tripolar mitosis is a key mechanism generating karyotype-wide aneuploidy in cleavage-stage embryos and implicate PLK4-mediated centrosome abnormality as a factor influencing its occurrence.

6 ANEUPLOIDY TOLERANCE IN RHESUS MACAQUE PRE-IMPLANTATION EMBRYOS VIA MICRONUCLEI FORMATION, CELLULAR FRAGMENTATION, AND BLASTOMERE EXCLUSION

2017 ◽  
Vol 29 (1) ◽  
pp. 110 ◽  
Author(s):  
B. L. Daughtry ◽  
J. L. Rosenkrantz ◽  
N. Lazar ◽  
N. Redmayne ◽  
K. A. Nevonen ◽  
...  
Keyword(s):  
Rhesus Macaque ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Chromosomal Mosaicism ◽  
Human Embryos ◽  
Cleavage Stage ◽  
Chromosomal Breakage

A primary contributor to in vitro fertilization (IVF) failure is the presence of unbalanced chromosomes in pre-implantation embryos. Previous array-based and next-generation sequencing (NGS) studies determined that ~50 to 80% of human embryos are aneuploid at the cleavage stage. During early mitotic divisions, many human embryos also sequester mis-segregated chromosomes into micronuclei and concurrently undergo cellular fragmentation. We hypothesised that cellular fragmentation represents a response to mis-segregated chromosomes that are encapsulated into micronuclei. Here, we utilised the rhesus macaque pre-implantation embryo as a model to study human embryonic aneuploidy using a combination of EevaTM time-lapse imaging for evaluating cell divisions, single-cell/-fragment DNA-Sequencing (DNA-Seq), and confocal microscopy of nuclear structures. Results from our time-lapse image analysis demonstrated that there are considerable differences in the timing of the first and third mitotic divisions between rhesus blastocysts and those that arrested before this stage in development (P < 0.01; ANOVA). By examining the chromosome content of each blastomere from cleavage stage embryos via DNA-Seq, we determined that rhesus embryos have an aneuploidy frequency up to ~62% (N = 26) with several embryos exhibiting chromosomal mosaicism between blastomeres (N = 6). Certain blastomeres also exhibited reciprocal whole chromosomal gains or losses, indicating that these embryos had undergone mitotic non-disjunction early in development. In addition, findings of reciprocal sub-chromosomal deletions/duplications among blastomeres suggest that chromosomal breakage had occurred in some embryos as well. Embryo immunostaining for the nuclear envelope protein, LAMIN-B1, demonstrated that fragmented cleavage-stage rhesus embryos often contain micronuclei and that cellular fragments can enclose DNA. Our DNA-Seq analysis confirmed that cellular fragments might encapsulate whole and/or partial chromosomes lost from blastomeres. When embryos were immunostained with gamma-H2AX, a marker of chromatin fragility, we observed distinct foci solely in micronuclei and DNA-containing cellular fragments. This suggests that micronuclei may be ejected from blastomeres through the process of cellular fragmentation and, once sequestered, these mis-segregated chromosomes become highly unstable and undergo DNA degradation. Finally, we also observed that ~10% of embryos prevented cellular fragments or large blastomeres from incorporating into the inner cell mass or trophectoderm at the blastocyst stage (n = 5). Upon confocal imaging, multiple nuclei and intense gamma-H2AX foci were found in a large unincorporated blastomere in one of the blastocysts. Altogether, our findings demonstrate that the rhesus embryo responds to segregation errors by eliminating chromosome-containing micronuclei via cellular fragmentation and/or selecting against aneuploid blastomeres that fail to divide during pre-implantation development with significant implications for human IVF.

scholarly journals Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage

Zygote ◽  
1994 ◽  
Vol 2 (4) ◽  
pp. 281-287 ◽  
Author(s):  
Asangla Ao ◽  
Robert P. Erickson ◽  
Robert M.L. Winston ◽  
Alan H Handysude
Keyword(s):  
Mammalian Species ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Cell Stage ◽  
Gonad Differentiation ◽  
Embryonic Genome ◽  
Human Preimplantation Embryos

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

Permanence of de novo segmental aneuploidy in sequential embryo biopsies

2020 ◽  
Vol 35 (4) ◽  
pp. 759-769
Author(s):  
M C Magli ◽  
C Albanese ◽  
A Crippa ◽  
G Terzuoli ◽  
G La Sala ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Initial Diagnosis ◽  
Preimplantation Embryos ◽  
Comparative Genomic ◽  
Cleavage Stage ◽  
Preimplantation Genetic Testing ◽  
Initial Finding ◽  
Segmental Aneuploidy ◽  
Chromosome Imbalance

Abstract STUDY QUESTION Is de novo segmental aneuploidy (SA) a biological event or an artifact that is erroneously interpreted as partial chromosome imbalance? SUMMARY ANSWER The detection of de novo SA in sequential biopsies of preimplantation embryos supports the biological nature of SA. WHAT IS KNOWN ALREADY Although some SAs are detected in oocytes and in blastocysts, the highest incidence is observed in cleavage-stage embryos. Based on these findings, we can postulate that the majority of cells affected by SAs are eliminated by apoptosis or that affected embryos mainly undergo developmental arrest. STUDY DESIGN, SIZE, DURATION This retrospective study includes 342 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between January 2014 and December 2018. Chromosome analysis was performed on 331 oocytes, 886 cleavage-stage embryos and 570 blastocysts (n = 1787). From 268 expanded blastocysts, the blastocoelic fluid (BF) was also analyzed (resulting in 2025 samples in total). In cases of SAs involving loss or gain in excess of 15 Mb, embryos were not considered for transfer and sequential biopsies were performed at following stages. This resulted in 66 sets where the initial diagnosis of SAs (4 made in polar bodies, 25 in blastomeres and 37 in trophectoderm (TE) cells) was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 2082 samples (2025 + 27 whole embryos) were processed by whole genome amplification followed by array comparative genomic hybridization. MAIN RESULTS AND THE ROLE OF CHANCE The incidence of SAs was 6.3% in oocytes, increased to 16.6% in cleavage-stage embryos (P &lt; 0.001) and decreased to 11.2% in blastocysts (P &lt; 0.025 versus oocytes; P &lt; 0.01 versus cleavage-stage embryos). The highest incidence of SAs was found in BFs (26.1%, P &lt; 0.001). The analysis of 66 sets of sequential biopsies revealed that the initial finding was confirmed in all following samples from 39 sets (59.1% full concordance). In 12 additional sets, SAs were detected in some samples while in others the interested chromosome had full aneuploidy (18.2%). In three more sets, there was a partial concordance with the initial diagnosis in some samples, but in all TE samples the interested chromosome was clearly euploid (4.5%). In the remaining 12 sets, the initial SA was not confirmed at any stage and the corresponding chromosomes were euploid (18.2% no concordance). The pattern of concordance was not affected by the number of SAs in the original biopsy (single, double or complex) or by the absence or presence of concomitant aneuploidies for full chromosomes. LIMITATIONS, REASONS FOR CAUTION Chromosome analyses were performed on biopsies that might not be representative of the true constitution of the embryo itself due to the occurrence of mosaicism. WIDER IMPLICATIONS OF THE FINDINGS The permanence of SAs throughout the following stages of embryo development in more than half of the analyzed sets suggests for this dataset a very early origin of this type of chromosome imbalance, either at meiosis or at the first mitotic divisions. Since SAs remained in full concordance with the initial diagnosis until the blastocyst stage, a corrective mechanism seems not to be in place. In the remaining cases, it is likely that, as for full chromosome aneuploidy, mosaicism derived from mitotic errors could have occurred. In following cell divisions, euploid cell lines could prevail preserving the embryo chances of implantation. Due to the scarcity of data available, the transfer of embryos with SAs should be strictly followed up to establish possible clinical consequences related to this condition. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was obtained. There are no conflicts of interest.

P–150 Does trophectoderm biopsy performed on different blastocyst stages affect the clinical outcome?

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S H Tan ◽  
A Q Y Chan ◽  
A Y X Lim ◽  
M W Lim
Keyword(s):  
Retrospective Study ◽  
Clinical Outcome ◽  
Sample Size ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Assisted Hatching ◽  
Preimplantation Genetic Testing ◽  
Significant Difference ◽  
Registration Number

Abstract Study question The objective of this study is to evaluate the effect of trophectoderm (TE) biopsy on different blastocyst stages and its clinical outcome. Summary answer Our results showed that TE biopsy significantly reduced the clinical outcome of fully hatched blastocyst. What is known already: TE biopsy is a method widely practiced to harvest cells to determine the chromosomal constitution of a blastocyst, ensuring higher implantation and healthy pregnancies. The effect on clinical outcome after transferring blastocysts biopsied at different blastocysts stages has not been extensively studied. Study design, size, duration This retrospective study was conducted from January 2017 until July 2019 at Alpha IVF & Women’s Specialists. Following laser assisted hatching on day 3, TE biopsy was performed on unhatched, hatching and fully hatched day–5 blastocysts. A total of 1,020 single euploid blastocysts transfer (SBT) were performed. The average maternal age was 31.7. Implantation rates (IR) were evaluated for all stages of hatching (Unhatched: BG3 & 4; hatching: BG5; fully hatched: BG6). Participants/materials, setting, methods Laser assisted hatching (Hamilton Thorne Bioscience, USA) was performed on day–3 and subsequently cultured to blastocyst-stage. Different hatching stages were observed using embryoscope time-lapse system (Vitrolife, Sweden) and were recorded. Day–5 blastocysts with at least BG3BB grade (Gardner’s System) were selected for TE biopsy and the biopsied cells were sent for preimplantation genetic testing for aneuploidy (PGT-A) using Next-Generation Sequencing (Life Technologies, USA). All blastocysts were vitrified and warmed using the Cryotec Method (Cryotech, Japan). Main results and the role of chance: All 1,020 blastocysts survived post-warmed (post-warm survival rate= 100%) and were transferred in frozen transfer cycles. TE biopsy performed on unhatched blastocysts showed a comparable IR to hatching blastocysts (60.0% [15/25] and 65.2% [627/961]). While fully hatched blastocysts (44.12% [15/34]) show a significantly lower IR when compared to hatching blastocysts (65.2% [627/961]), no significant difference was seen when comparing unhatched blastocysts to fully hatched blastocysts (60.0% [15/25] and 44.12% [15/34]; p = 0.2949). Limitations, reasons for caution The sample size was comparatively smaller in the unhatched and fully hatched group than the hatching group. Further studies with a larger sample size is recommended to ascertain the clinical outcome. Since this is a retrospective study and biopsy was done by different embryologists, the biopsy technique was not controlled. Wider implications of the findings: To achieve higher clinical pregnancy, it is recommended to perform TE biopsy before the blastocysts is fully hatched. Trial registration number Not applicable

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