scholarly journals Perivitelline threads in cleavage-stage human embryos: observations using time-lapse imaging

2017 ◽  
Vol 35 (6) ◽  
pp. 646-656 ◽  
Author(s):  
Louise Kellam ◽  
Laura M. Pastorelli ◽  
Angel M. Bastida ◽  
Amy Senkbeil ◽  
Sue Montgomery ◽  
...  
Keyword(s):  
Time Lapse ◽  
Human Embryos ◽  
Cleavage Stage ◽  
Time Lapse Imaging

Cleavage of Human Embryos: Options and Diversity

Acta Naturae ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 88-96
Author(s):  
Yu. K. Doronin ◽  
I. V. Senechkin ◽  
L. V. Hilkevich ◽  
M. A. Kurcer
Keyword(s):  
Time Lapse ◽  
Reproductive Technologies ◽  
Human Embryos ◽  
Imaging Data ◽  
Noninvasive Methods ◽  
Topographic Features ◽  
Time Parameters ◽  
Embryo Cleavage ◽  
Time Lapse Imaging ◽  
Developmental Dynamics

In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

P–201 The beneficial effects of ZP-free culture on cytoplasmic fragmentation in human embryos. : An innovative trial using 3PN zygotes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Sugishima ◽  
K Yumoto ◽  
T Shimura ◽  
Y Mio
Keyword(s):  
Embryo Development ◽  
Imaging System ◽  
Culture Method ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Human Embryos ◽  
Beneficial Effects ◽  
Molecular Biological Analysis ◽  
A Prospective Study ◽  
Time Lapse Imaging

Abstract Study question Is it possible to culture ZP-free embryos to eliminate perivitelline threads, which are known to be involved in generating cytoplasmic fragments at the first cleavage? Summary answer ZP-free culturing, an innovative system that decreases the amount of cytoplasmic fragments without disrupting the blastomeres, using incubators with time-lapse imaging. What is known already A study in 2017 observed perivitelline threads in more than 50% of cleavage-stage human embryos using time-lapse imaging, and the rate of cytoplasmic fragmentation (at the first cleavage) was significantly decreased in embryos without perivitelline threads (P < 0.001). While it has been proposed that perivitelline threads play an important role in crosslinking the cumulus cells and oocyte during maturation, the mechanism underlying such a role remains unclear. It is also unknown whether the threads still function in mature MII oocytes. Study design, size, duration A prospective study was conducted using 2,852 normal (2PN/2PB) embryos from c-IVF/ICSI and 113 abnormal (3PN) embryos obtained from c-IVF between 2017 and 2019. The zona pellucida (ZP) of 71 abnormal embryos was removed at the pronuclear stage (“ZP-free”), and the rest (n = 42) were cultured as “ZP-intact”. Normal and abnormal embryos were cultured for five days in bench-top incubators (MINC, COOK) and an incubator equipped with a time-lapse imaging system. Participants/materials, setting, methods Embryos used in this study were donated by 412 couples who underwent c-IVF cycles in our clinic between 2017 and 2019. For ZP removal, 3PN embryos were placed in 0.125M sucrose-containing HEPES media drops to reduce the ooplasm size. Then, ooplasms were completely separated from ZPs by a laser and pipetting. Embryo development and morphology of the three groups (normal, ZP-intact and ZP-free abnormal) were compared based on the degree of cytoplasmic fragmentation. Main results and the role of chance The first cleavage occurred in 97.8% (n = 2,790/2,852) of 2PN/2PB, 83.3% (n = 35/42) of ZP-intact 3PN and 97.2% (n = 69/71) of ZP-free 3PN. Normal (2PN/2PB), ZP-intact and ZP-free 3PN embryos were classified into three groups based on the modified Veeck’s criteria thus: <20% fragmented compared to the total volume of cytoplasm at the first cleavage (Grade 1 and 2, Good); 20–39% fragmented (Grade 3, Fair) and ≧40% fragmented (Grade 4, Poor). Of 69 cleaved ZP-free 3PN embryos, 68.1% (n = 47) showed less than 20% fragments which was significantly higher than 2PN/2PB (43.7%, n = 1,218/2,790) and ZP-intact 3PN (45.7%, n = 16/35; P < 0.05). Furthermore, 24.6% (n = 17/69) of ZP-free 3PN embryos showed 20–39% fragments which was significantly lower than 2PN/2PB (45.9%, n = 1,281/2,790; P < 0.05). In addition, 50.7% of ZP-free 3PN embryos (n = 36) developed to the morula stage after the third cleavage, and 29.6% (n = 21) formed blastocoel and became blastocysts. Thus, removing the ZP before the first cleavage did not adversely affect embryo development and decreased the cytoplasmic fragmentation. Limitations, reasons for caution Due to ethical and clinical limitations, we only examined abnormally fertilized embryos in this study. Moreover, since the relationship between the perivitelline threads and cytoplasmic fragments is unclear, we plan to conduct molecular biological analysis of the perivitelline threads in further studies. Wider implications of the findings: This study revealed that ZP is not always necessary after the pronuclear stage because ZP-free embryos studied herein developed normally and maintained cell adhesion well. This innovative culture method might provide the breakthrough needed for patients to improve embryo quality who obtain embryos with severe fragmentation caused by perivitelline threads. Trial registration number Not applicable

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 Morphokinetic analysis of cleavage stage embryos and its relationship to aneuploidy in a retrospective time-lapse imaging study

2014 ◽  
Vol 32 (1) ◽  
pp. 69-75 ◽  
Author(s):  
Monika Chawla ◽  
Michael Fakih ◽  
Amal Shunnar ◽  
Asina Bayram ◽  
Ali Hellani ◽  
...  
Keyword(s):  
Imaging Study ◽  
Time Lapse ◽  
Cleavage Stage ◽  
Time Lapse Imaging

scholarly journals Comparing transcriptome profiles of human embryo cultured in closed and standard incubators

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9738
Author(s):  
Jingyu Li ◽  
Jiayu Huang ◽  
Wei Han ◽  
Xiaoli Shen ◽  
Ying Gao ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Positive Impact ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Enrichment Analysis ◽  
Time Lapse ◽  
Human Embryos ◽  
Rna Seq ◽  
False Discovery ◽  
Time Lapse Imaging

It is necessary to compare the transcriptomic profiles of human embryos cultured in time-lapse imaging (TLI) incubators and standard incubators (SI) in order to determine whether a closed culture system has a positive impact on embryos. In this study, we used RNA-sequencing (RNA-Seq) to characterize and compare the gene expression profiles of eight-cell embryos of the same quality grade cultured in TLI and SI. We sequenced a total of 580,952,620 reads for zygotes, TLI-cultured, and SI-cultured eight-cell embryos. The global transcriptomic profiles of the TLI embryos were similar to those of the SI embryos and were highly distinct from the zygotes. We also detected 539 genes showing differential expression between the TLI and SI groups with a false discovery rate (FDR) < 0.05. Using gene ontology enrichment analysis, we found that the highly expressed SI genes tended to execute functions such as transcription, RNA splicing, and DNA repair, and that the highly expressed TLI genes were enriched in the cell differentiation and methyltransferase activity pathways. This study, the first to use transcriptome analysis to compare SI and TLI, will serve as a basis for assessing the safety of TLI application in assisted reproductive technology.

151 Multipolar zygotic divisions result in multinuclear and anuclear blastomeres in cattle

2021 ◽  
Vol 33 (2) ◽  
pp. 184
Author(s):  
T. De Coster ◽  
K. Smits ◽  
O. B. Pascottini ◽  
J. Vermeesch ◽  
A. Van Soom
Keyword(s):  
Time Lapse ◽  
Human Embryos ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Mixed Effect ◽  
Genetic Abnormalities ◽  
The Individual ◽  
Time Lapse Imaging ◽  
Nuclear Content

The mammalian zygotic cleavage is expected to result in two mononuclear blastomeres. However, zygotes undergoing multipolar divisions resulting in direct cleavage into three or four cells are frequently observed in bovine and human embryonic development and have been associated with decreased euploidy rates of resulting blastocysts and a lower pregnancy rate (Somfai et al. 2010 J. Reprod. Dev. 56, 200-207; https://doi.org/10.1262/jrd.09-097a; Zhan et al. 2016 PLoS ONE 11, 1-19; https://doi.org/10.1371/journal.pone.0166398; Sugimura et al. 2017 J. Reprod. Dev. 63, 353-357; https://doi.org/10.1262/jrd.2017-041). Therefore, multipolar zygotic divisions may underly genetic abnormalities by aberrant segregation of the chromosomal material resulting in multinucleated or anuclear blastomeres. These abnormal blastomeres have been observed in human cleavage-stage embryos (Nogueira et al. 2000 Fertil. Steril. 74, 295-298; https://doi.org/10.1016/s0015-0282(00)00642-7; Chatzimeletiou et al. 2006 Hum. Reprod. 20, 672–682; https://doi.org/10.1093/humrep/deh652), but the prevalence in bovine embryos and the direct association with the multipolar division in both bovine and human embryos remains unknown. We hypothesised that anuclear and multinuclear blastomeres also occur in bovine embryos, and we aimed to unravel the link between multipolar zygotic divisions and genome segregation errors by determining the nuclear blastomere content in a bovine model. Therefore, oocytes from 5 cows were matured and fertilized in vitro by the same bull according to our standard in vitro production procedure (Wydooghe et al. 2014 Reproduction 148, 519-529). The first cleavage was monitored the by time-lapse imaging. Forty-three blastomeres from 22 bipolar zygotic divisions, and 65 blastomeres from 20 multipolar zygotic divisions were collected immediately after the first cleavage, using pronase to isolate the individual blastomeres. The area of each blastomere was measured and the number of nuclei was determined after fixation and staining with Hoechst 33342. Generalized mixed effect models were built to identify the effect of the type of cleavage (bipolar vs. multipolar) on the number of nuclei (mononuclear vs. anuclear or multinuclear) in the blastomeres. Linear mixed models were built to determine the effect of the type of cleavage and the nuclear content on the size of the blastomeres. Embryos presented a greater number of blastomeres with a normal nuclear content (92.6 ± 0.4%) after a bipolar cleavage compared with multipolar division (73.2 ± 0.7%; P=0.03). Moreover, blastomeres presented a 28% larger blastomere area (P&lt;0.001) after bipolar division compared with multipolar division. Notably, anuclear blastomeres tended to be smaller than multi- and mononuclear blastomeres (P=0.09 for both), while no difference was found between mono and multinucleated blastomeres (P=0.84). In conclusion, this is one of the first reports on the association between nuclear blastomere content in bovine embryos and the dynamics of the first zygotic division. Even though sample size was limited, these results confirm the hypothesised link between multipolar division and abnormal genome segregation as determined by multinuclear and anuclear blastomeres in the resulting blastomeres. Therefore, multipolar cell divisions at the zygotic division may underly at least some of the genetic abnormalities observed in embryos at early development.

Time-lapse imaging cleavage stage parameters do not enhance selection among traditionally selected high quality euploid blastocysts that implant

2014 ◽  
Vol 102 (3) ◽  
pp. e304-e305
Author(s):  
C. Juneau ◽  
K.H. Hong ◽  
M.D. Werner ◽  
J.M. Franasiak ◽  
K.M. Upham ◽  
...  
Keyword(s):  
Time Lapse ◽  
Cleavage Stage ◽  
High Quality ◽  
Time Lapse Imaging
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