O-003 The puzzling unknowns of abnormal fertilization and first cleavage

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Racowsky
Keyword(s):  
Embryo Development ◽  
Time Lapse ◽  
Critical Event ◽  
Cleavage Division ◽  
Sequence Of Events ◽  
Polar Bodies ◽  
Abnormal Fertilization ◽  
Temporal And Spatial ◽  
Temporal Events ◽  
Time Lapse Imaging

Abstract text Fertilization is a critical event in development in that it provides the connection between the gametes and the earliest stages of embryogenesis. Yet, despite the central importance of this process in contributing to embryo developmental fate, clinical embryologists have historically assessed fertilization merely by the number of pronuclei and, if two are present, perhaps, by the presence of two polar bodies. Even though over 20 years ago, time lapse imaging was applied for defining early events of fertilization (Payne et al., 1997), it is only with contemporary time-lapse imaging systems in the last few years that detailed evaluation of spatial and temporal events of fertilization have been described (Iwata & Yasuyuki, 2016; Cottichio et al., 2018). These careful analyses allow us to describe typical and atypical events of fertilization and how they are each associated with timing of the first cleavage division and subsequent embryo development. In this lecture, we will first describe the fundamental underpinnings of fertilization and highlight the normal events associated with this process. We will then discuss gross morphological abnormalities as visualized by light microscopy and highlight the unknowns associated with these events. Finally, we will focus on time-lapse imaging studies, which have revealed the remarkable spatial and temporal coordination of meiotic resumption, pronuclear dynamics, chromatin organization and cytoplasmic/cortical modifications that occur during fertilization and the implications of aberrations for the first cleavage division. At the conclusion of this presentation, attendees should be able to: Review the normal events associated with fertilization and the first cleavage division. 1 Describe gross morphological aberrations of these two fundamental processes. 2 Discuss temporal and spatial abnormalities in the coordinated sequence of events that underly these processes. 3 State the potential application of these abnormalities as predictors of abnormal embryo development. 4 Summarize the puzzling unknowns that underly these abnormalities.

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

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

Time-lapse imaging: the state of the art†

2019 ◽  
Vol 101 (6) ◽  
pp. 1146-1154 ◽  
Author(s):  
Raquel Del Gallego ◽  
José Remohí ◽  
Marcos Meseguer
Keyword(s):  
Embryo Development ◽  
Clinical Success ◽  
Time Lapse ◽  
Embryo Cell ◽  
Cell Dynamics ◽  
Vitro Fertilization ◽  
Cycle Lengths ◽  
Novel Concept ◽  
Time Lapse Imaging

Abstract The introduction of time-lapse imaging to clinical in vitro fertilization practice enabled the undisturbed monitoring of embryos throughout the entire culture period. Initially, the main objective was to achieve a better embryo development. However, this technology also provided an insight into the novel concept of morphokinetics, parameters regarding embryo cell dynamics. The vast amount of data obtained defined the optimal ranges in the cell-cycle lengths at different stages of embryo development. This added valuable information to embryo assessment prior to transfer. Kinetic markers became part of embryo evaluation strategies with the potential to increase the chances of clinical success. However, none of them has been established as an international standard. The present work aims at describing new approaches into time-lapse: progress to date, challenges, and possible future directions.

P–212 Mitochondrial DNA content shows a significant association with timing of human embryo development and fertility diagnosis in euploid embryos

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Hur ◽  
V Nanavaty ◽  
A Chehab ◽  
M Yao ◽  
N Desai
Keyword(s):  
Mitochondrial Dna ◽  
Embryo Development ◽  
Dna Content ◽  
Time Lapse ◽  
Correlation Factor ◽  
Embryo Selection ◽  
Preimplantation Genetic Testing ◽  
Additional Tool ◽  
Mitochondrial Dna Content ◽  
Time Lapse Imaging

Abstract Study question Does mitochondrial DNA content (mtDNA) correlate with clinical parameters and embryo morphokinetics using advanced time-lapse technology? Summary answer mtDNA correlated with embryo morphokinetics and the growth trajectory of euploid embryos. Maternal age, anti-mullerian hormone level and fertility diagnosis were significantly associated with mtDNA. What is known already With the push towards single embryo transfers, laboratories are working to improve embryo selection. In addition to conventional microscopy, preimplantation genetic testing and time-lapse microscopy have been utilized to aid in embryo selection. More recently, as mtDNA may represent the energy potential of an embryo, some data have supported the use of mtDNA as an additional tool. Limited studies have suggested that a lower amount of mtDNA is associated with higher rates of implantation and improved embryo quality. Study design, size, duration This is a retrospective chart review. All embryos that underwent preimplantation genetic testing for aneuploidy (PGT-A) between January to December of 2020 were studied. Participants/materials, setting, methods Women undergoing in vitro fertilization (IVF) with intracytoplasmic sperm injection undergoing PGT-A were studied. All patients were from a single academic institution. This study exclusively examined the characteristics of euploid embryos. Mitochondrial DNA content was expressed as a ratio of mtDNA:nDNA (MitoScore). Time-lapse imaging was utilized to evaluate embryo development every 15 minutes in 5–7 focal planes. Chi square test and Spearman correlation analysis were performed with a p-value of < 0.05 considered significant. Main results and the role of chance A total of 494 embryos from 52 women who underwent 58 IVF cycles were cultured to blastocyst and 331 embryos were biopsied for PGT-A evaluation. Of these, 132 embryos were diagnosed as euploid. A moderate positive correlation was found between MitoScore and time to morula, time to blast and time to expanded blast (correlation value 0.54, 0.50 and 0.54, respectively; p < 0.001). Consistent with this trend, day 5 blastocysts had a significantly lower MitoScore values than day 6 blastocysts (20.2 v. 29.2; p < 0.001). When examining all biopsied euploid embryos, no significant association was found between MitoScore, blastocyst maturity, trophectoderm or inner cell mass scores. Our data also demonstrated a positive correlation between MitoScore and maternal age (correlation factor 0.33; p < 0.001). A negative association between MitoScore and serum anti-mullerian hormone levels (correlation factor –0.20; p < 0.021) was also noted. Of particular interest was the significant association between fertility diagnosis and mitochondrial score (p < 0.001). Even amongst euploid embryos, mtDNA content varied widely, potentially reflecting differences in embryo potential and quality. Additionally, the significant difference in MitoScore between that day 5 and day 6 blastocysts may reflect a fundamental difference in cytoplasmic characteristics and requires further study. Limitations, reasons for caution Due to the study cohort of euploid embryos undergoing PGT-A, this study was biased for the selection of high grade embryos. This limited diversity in embryo quality may have masked other potential associations between mitochondrial content and blastocyst quality. Wider implications of the findings: mtDNA may be additional tool aiding in embryo selection as IVF labs work to improve pregnancy rates while minimizing the risks of transferring multiple embryos. To our knowledge, this is the largest study assessing the relationship of mtDNA content of blastocysts and the timing of embryo development using time-lapse imaging. Trial registration number None

Preliminary morphokinetic annotation data for early equine embryo development in vitro using a time-lapse imaging system

2016 ◽  
Vol 41 ◽  
pp. 72
Author(s):  
Karen Schnauffer ◽  
Niamh Lewis ◽  
Stephen Troup ◽  
Dai Grove-White ◽  
Caroline McG. Argo
Keyword(s):  
Embryo Development ◽  
Imaging System ◽  
Time Lapse ◽  
Annotation Data ◽  
Development In Vitro ◽  
Time Lapse Imaging

Temporal and Spatial Dynamics of the Actin-Based Cytoskeleton

1990 ◽  
Vol 48 (2) ◽  
pp. 546-546
Author(s):  
D. Lansing Taylor
Keyword(s):  
Spatial Dynamics ◽  
Time Lapse ◽  
Living Cells ◽  
Biomedical Sciences ◽  
3T3 Cells ◽  
Cellular Functions ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Temporal And Spatial ◽  
Time Lapse Imaging

There has been a renaissance and revolution in the use of light microscopy in the biomedical sciences. The renaissance has been due to the importance of studying the temporal and spatial dynamics of ions, metabolites and macromolecules in living cells and tissues. The revolution has been due to the integration of developments in molecular biology, fluorescent probe chemistry, machine vision, and imaging technology. It is now possible to use the living cell as a microcuvette and to explore the chemical and molecular dynamics responsible for cellular functions.We have been investigating the formation, transport and contraction of stress fibers in Swiss 3T3 cells. Fluorescent analogs of actin, myosin, vinculin and profilin have been investigated in serum deprived cells before, during and after stimulation with thrombin. The activities of these components of the actin-based cytoskeleton have been quantified using time-lapse imaging, fluorescence redistribution after photobleaching, video-enhanced contrast and reflection interference contrast microscopy.

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