scholarly journals Prediction of the in vitro developmental competence of early-cleavage-stage human embryos with time-lapse imaging and oxygen consumption rate measurement

2018 ◽  
Vol 17 (3) ◽  
pp. 289-296 ◽  
Author(s):  
Kaori Goto ◽  
Yoko Kumasako ◽  
Megumi Koike ◽  
Akiko Kanda ◽  
Kyoko Kido ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Consumption Rate ◽  
Time Lapse ◽  
Developmental Competence ◽  
Human Embryos ◽  
Rate Measurement ◽  
Cleavage Stage ◽  
Early Cleavage ◽  
Time Lapse Imaging

Prediction of in-vitro developmental competence of early cleavage-stage mouse embryos with compact time-lapse equipment

2010 ◽  
Vol 20 (3) ◽  
pp. 371-379 ◽  
Author(s):  
Csaba Pribenszky ◽  
Eszter Losonczi ◽  
Miklós Molnár ◽  
Zsolt Lang ◽  
Szabolcs Mátyás ◽  
...  
Keyword(s):  
Time Lapse ◽  
Mouse Embryos ◽  
Developmental Competence ◽  
Cleavage Stage ◽  
Early Cleavage

Morphokinetics of early equine embryo development in vitro using time-lapse imaging, and use in selecting blastocysts for transfer

2019 ◽  
Vol 31 (12) ◽  
pp. 1851 ◽  
Author(s):  
Niamh Lewis ◽  
Karen Schnauffer ◽  
Katrin Hinrichs ◽  
Monica Morganti ◽  
Stephen Troup ◽  
...  
Keyword(s):  
Embryo Development ◽  
Time Lapse ◽  
Developmental Competence ◽  
Early Cleavage ◽  
Blastocyst Development ◽  
Use Of Time ◽  
Development In Vitro ◽  
Time Lapse Imaging ◽  
First Time

The use of time-lapse imaging (TLI) in the evaluation of morphokinetics associated with invitro developmental competence is well described for human, cattle and pig embryos. It is generally accepted that embryos that complete early cleavage sooner are more likely to form blastocysts and that timing of later events, such as blastocyst formation and expansion, are predictive of implantation potential and euploid status. In the horse, morphokinetics as a predictor of developmental competence has received little attention. In this study we evaluated the morphokinetics of early equine embryo development invitro for 144 oocytes after intracytoplasmic sperm injection and report the timings of blastocyst development associated with ongoing pregnancy for the first time. There was a tendency for time of cytoplasmic extrusion and first cleavage to occur earlier in the embryos that went on to form blastocysts (n=19) compared with those that arrested, and for first cleavage to occur earlier in blastocysts that established pregnancies that were ongoing (n=4) compared with pregnancies that were lost (n=2). TLI was clinically useful in identifying blastocysts when evaluation of morphology on static imaging was equivocal.

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 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

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.

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.

scholarly journals Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

2021 ◽  
Vol 22 (16) ◽  
pp. 8367
Author(s):  
Hien Lau ◽  
Shiri Li ◽  
Nicole Corrales ◽  
Samuel Rodriguez ◽  
Mohammadreza Mohammadi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Oxygen Consumption Rate ◽  
Consumption Rate ◽  
In Vitro Development ◽  
Porcine Islets ◽  
Vitro Development

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

Growth and viability of human blastocysts in vitro

2000 ◽  
Vol 8 (3) ◽  
pp. 241-287 ◽  
Author(s):  
GM Jones
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Early Cleavage ◽  
Uterine Endometrium ◽  
Stage Of Development ◽  
Uterine Lavage

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

scholarly journals Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Methods ◽  
2018 ◽  
Vol 133 ◽  
pp. 81-90 ◽  
Author(s):  
Katja M. Piltti ◽  
Brian J. Cummings ◽  
Krystal Carta ◽  
Ayla Manughian-Peter ◽  
Colleen L. Worne ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Neural Stem Cells ◽  
Single Cell ◽  
Cell Tracking ◽  
Time Lapse ◽  
Live Cell ◽  
Time Lapse Imaging
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