Blastocyst culture: analysis of factors affecting the development of embryos to the blastocyst stage

The rhesus macaque (Macaca mulatta) is widely used for the study of human disease. Increasingly assisted reproductive techniques are applied to the propagation of this species to identify specific genotypes, to generate animals with genetic modifications, or to preserve valuable genetic resources. A significant advantage in this endeavor would be the ability to identify embryos with desired genetic characteristics by pre-implantation genetic diagnosis (PGD) prior to their transfer into a surrogate. The objectives of this study, therefore, were to assess the feasibility of performing blastomere biopsies on rhesus embryos and to determine the effects of (a) oxygen tension during culture, (b) transient cryopreservation, and (c) the number of blastomeres removed (one or two) on subsequent development. A total of 17 oocyte aspirations were performed, which resulted in 206 8- to 10-cell embryos that were selected for biopsy. Embryos were cultured in HECM-9 medium and cryopreserved with propylene glycol. The chi-square test was used for data analysis. For the first experiment, 160 embryos were used to assess the effect of oxygen tension and transient cryopreservation. Oxygen tension during culture had no effect on the number of embryos proceeding to the blastocyst stage (26.0 vs. 25.0%) following blastomere removal. Similarly, transient freezing and storage in liquid nitrogen did not affect subsequent development to the blastocyst stage (26.6 vs. 24.7%). For the second experiment, 46 embryos were biopsied and assigned to removal of either one or two blastomeres. Subsequent development was not affected by the number of blastomeres removed, and a similar percentage of embryos reached the blastocyst stage (38.1 vs. 20%). These results demonstrate that rhesus embryos are tolerant of biopsies and proceed to advanced stages of development at the same rate as control embryos. Funding was provided by the Tulane National Primate Research Center base grant NIH 5P51 RR00164-44.

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Mouse embryo motion and embryonic development from the 2-cell to blastocyst stage using mechanical vibration systems

Reproduction Fertility and Development ◽

10.1071/rd13039 ◽

2014 ◽

Vol 26 (5) ◽

pp. 733 ◽

Cited By ~ 4

Author(s):

Yuka Asano ◽

Koji Matsuura

Keyword(s):

Embryonic Development ◽

Mechanical Vibration ◽

Blastocyst Stage ◽

Mouse Embryos ◽

Physical Factors ◽

Mechanical Stimuli ◽

Negative Effects ◽

Factors Affecting ◽

Comparison Of The Results ◽

Static Conditions

We investigated the effect of mechanical stimuli on mouse embryonic development from the 2-cell to blastocyst stage to evaluate physical factors affecting embryonic development. Shear stress (SS) applied to embryos using two mechanical vibration systems (MVSs) was calculated by observing microscopic images of moving embryos during mechanical vibration (MV). The MVSs did not induce any motion of the medium and the diffusion rate using MVSs was the same as that under static conditions. Three days of culture using MVS did not improve embryonic development. MVS transmitted MV power more efficiently to embryos than other systems and resulted in a significant decrease in development to the morula or blastocyst stage after 2 days. Comparison of the results of embryo culture using dynamic culture systems demonstrated that macroscopic diffusion of secreted materials contributes to improved development of mouse embryos to the blastocyst stage. These results also suggest that the threshold of SS and MV to induce negative effects for mouse embryos at stages earlier than the blastocyst may be lower than that for the blastocyst, and that mouse embryos are more sensitive to physical and chemical stimuli than human or pig embryos because of their thinner zona pellucida.

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Value of transferring embryos derived from monopronucleated (1PN) zygotes at the time of fertilization assessment

Zygote ◽

10.1017/s096719942000009x ◽

2020 ◽

Vol 28 (3) ◽

pp. 241-246 ◽

Cited By ~ 2

Author(s):

Ming Li ◽

Yujiao Dang ◽

Ying Wang ◽

Junsheng Li ◽

Ping Liu

Keyword(s):

Pregnancy Rate ◽

Retrospective Analysis ◽

Intracytoplasmic Sperm Injection ◽

Patient Characteristics ◽

Blastocyst Stage ◽

Cleavage Stage ◽

Blastocyst Culture ◽

Live Birth Rates ◽

Comparison Groups

SummaryThis paper is a retrospective analysis of the sole transfer of monopronucleated zygotes (1PN) embryos both in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to determine the value of transferring embryos formed from 1PN. In fresh cycles, 1PN cleavage-stage embryos (1PN cleavage fresh) were transferred. In frozen–thawed cycles, 1PN blastocyst-stage embryos (1PN blast frozen) were transferred. We used comparison groups: for fresh cycles, 2PN cleavage-stage embryos (2PN cleavage fresh) were transferred; and for frozen–thawed cycles, 2PN blastocyst-stage embryos (2PN blast frozen) were transferred. Comparison groups were matched for cycle and patient characteristics to the 1PN group. Finally, for fresh cycles, live birth rates (LBR) in the 1PN cleavage group were significantly lower than those in 2PN cleavage group, both for IVF [LBR = 7.64% vs. pregnancy rate (PR) = 22.12%, P = 0.003, respectively] and ICSI (LBR = 0% vs. LBR = 20.00%, P < 0.001, respectively). For frozen–thawed IVF cycles, the PR in the 1PN blastocyst group were comparable with those of the 2PN blastocyst group (1PN: LBR = 33.14% vs. 2PN: LBR = 37.24%, P = 0.289, respectively), while in ICSI, the PR in the 1PN blastocyst group were lower than those in the 2PN blastocyst group (LBR = 15.25% vs. LBR = 40.68%, P = 0.002, respectively). So, for IVF, blastocyst culture was capable of selecting normal 1PN embryos for transfer and achieves satisfying outcomes. However, for ICSI, blastocyst culture was not effective enough to eliminate abnormal embryos and 1PN embryo transfer needed to be treated with caution.

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In vitro culture of embryos produced by in vitro maturation and fertilization of bovine follicular oocytes

Proceedings of the British Society of Animal Production (1972) ◽

10.1017/s0308229600010151 ◽

1989 ◽

Vol 1989 ◽

pp. 14-14

Author(s):

V. Vergos ◽

A. Gordon ◽

M. Gallagher ◽

I. Gordon

Keyword(s):

In Vitro Maturation ◽

Present Report ◽

Subsequent Development ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Fertilization In Vitro ◽

Factors Affecting ◽

Stage Of Development

A previous report from this laboratory dealt with the establishment of pregnancies in the early months of gestation after the non-surgical transfer of cattle embryos derived from the in vitro maturation (IVM) of primary bovine oocytes, their fertilization in vitro (IVF) and their subsequent development to the transferable stage (morula/blastocyst) using an in vivo (sheep oviduct) culture system (Lu et al.,1987). The present report deals with some factors affecting the efficiency of IVF and with the culture in vitro of zygotes to the morula/ blastocyst stage of development. Some embryos were frozen and after thawing transferred by non-surgical procedures to five recipient cattle to obtain information on their capacity to undergo further embryonic development.Primary oocytes, enclosed in cumulus cells, were recovered from vesicular follicles (2-6mm) after their dissection from the ovaries of heifers slaughtered at a local abattoir. The ovaries were brought to the laboratory within one hour of animal slaughter in medium held at 35'C.

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Progress In The Practical Application Of Automated Image Analysis To Tem Negatives

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100078614 ◽

1977 ◽

Vol 35 ◽

pp. 214-217

Author(s):

F. A. Heckman ◽

E. Redman ◽

J.E. Connolly

Keyword(s):

Image Analysis ◽

Image Quality ◽

Exposure Time ◽

Image Contrast ◽

Automated Image Analysis ◽

Practical Application ◽

Factors Affecting ◽

High Image Quality ◽

Qualitative Evidence ◽

Comprehensive Study

In our initial publication on this subject1) we reported results demonstrating that contrast is the most important factor in producing the high image quality required for reliable image analysis. We also listed the factors which enhance contrast in order of the experimentally determined magnitude of their effect. The two most powerful factors affecting image contrast attainable with sheet film are beam intensity and KV. At that time we had only qualitative evidence for the ranking of enhancing factors. Later we carried out the densitometric measurements which led to the results outlined below.Meaningful evaluations of the cause-effect relationships among the considerable number of variables in preparing EM negatives depend on doing things in a systematic way, varying only one parameter at a time. Unless otherwise noted, we adhered to the following procedure evolved during our comprehensive study:Philips EM-300; 30μ objective aperature; magnification 7000- 12000X, exposure time 1 second, anti-contamination device operating.

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3-D examination of the cytoskeletal sheets of mammalian eggs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124975 ◽

1992 ◽

Vol 50 (1) ◽

pp. 914-915

Author(s):

Carolyn A. Larabell ◽

David G. Capco ◽

G. Ian Gallicano ◽

Robert W. McGaughey ◽

Karsten Dierksen ◽

...

Keyword(s):

Actin Filaments ◽

Somatic Cells ◽

Freeze Fracture ◽

Blastocyst Stage ◽

Cell Cytoplasm ◽

Planar Structures ◽

Cytoskeletal Network ◽

Entire Cell ◽

Cytoskeletal Networks ◽

Mammalian Eggs

Mammalian eggs and embryos contain an elaborate cytoskeletal network of “sheets” which are distributed throughout the entire cell cytoplasm. Cytoskeletal sheets are long, planar structures unlike the cytoskeletal networks typical of somatic cells (actin filaments, microtubules, and intermediate filaments), which are filamentous. These sheets are not found in mammalian somatic cells nor are they found in nonmammalian eggs or embryos. Evidence that they are, indeed, cytoskeletal in nature is derived from studies demonstrating that 1) the sheets are retained in the detergent-resistant cytoskeleton fraction; 2) there are no associated membranes (determined by freeze-fracture); and 3) the sheets dissociate into filaments at the blastocyst stage of embryogenesis. Embedment-free sections of hamster eggs viewed at 60 kV show sheets running across the egg cytoplasm (Fig. 1). Although this approach provides excellent global views of the sheets and their reorganization during development, the mechanism of image formation for embedment-free sections does not permit evaluation of the sheets at high resolution.

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Cell-matrix interactions in the early mouse embryo

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123404 ◽

1992 ◽

Vol 50 (1) ◽

pp. 600-601

Author(s):

A.E. Sutherland ◽

P.G. Calarco ◽

C.H. Damsky

Keyword(s):

Mouse Embryo ◽

Giant Cells ◽

Blastocyst Stage ◽

Integrin Subunit ◽

Β1 Integrin ◽

Cell Stage ◽

Early Mouse Embryo ◽

Migratory Activity ◽

Early Mouse ◽

Cell Matrix Interactions

Cell-extracellular matrix (ECM) interactions mediated by the integrin family of receptors are critical for morphogenesis and may also play a regulatory role in differentiation during early development. We have examined the onset of expression of individual integrin subunit proteins in the early mouse embryo, and their roles in early morphogenetic events. As detected by immunoprecipitation, the α6, αV, β1, and β3 subunits are detected as early as the 4-cell stage, α5 at the hatched blastocyst stage and αl and α3 following blastocyst attachment. We tested the role of these integrins in the attachment and migratory activity of two cell populations of the early mouse embryo: the trophoblast giant cells, which invade the uterine stroma and ultimately contribute to the chorio-allantoic placenta, and the parietal endoderm, which migrates over the inner surface of the trophoblast and ultimately forms Reichert's membrane and the parietal yolk sac. Experiments were done in serum-free medium on substrates coated with laminin (Ln) and fibronectin (Fn). Trophoblast outgrowth occurs on Ln and its E8 fragment (long arm), but not on the E1’ fragment (cross region) (Figs. 1, 2 ). This outgrowth is inhibited by anti-E8, anti-Ln, and by the anti-β1 family antiserum anti-ECMR, but not by anti-αV or the function-perturbing GoH3 antibody that recognizes the α6/β1 integrin, a major Ln (E8) receptor. This suggests that trophoblast outgrowth on Ln or E8 is mediated by a different β1 integrin such as α3/β1. Early stages of trophoblast outgrowth (up to 48 hours) on Fn are inhibited by anti-Fn and by function-perturbing anti-αV antibodies, whereas at later times outgrowth becomes insensitive to anti-αV but remains sensitive to the anti-β1 family antiserum anti-ECMr, indicating that trophoblast cells modulate their interaction with Fn during outgrowth. Trophoblast outgrowth on vitronectin (Vn) is sensitive to anti-αV antibodies throughout the 5-day period examined.

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Factors affecting microstructural scale in liquid crystalline polymers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100174230 ◽

1990 ◽

Vol 48 (4) ◽

pp. 220-221

Author(s):

Christine M. Dannels ◽

Christopher Viney

Keyword(s):

Liquid Crystalline ◽

Liquid Crystalline Polymers ◽

Factors Affecting ◽

Crystalline Polymers ◽

Thermal Expansion Coefficients ◽

Low Thermal Expansion ◽

Lower Viscosity ◽

Fine Microstructure ◽

Planar Orientation ◽

Strength And Stiffness

Processing polymers from the liquid crystalline state offers several advantages compared to processing from conventional fluids. These include: better axial strength and stiffness in fibers, better planar orientation in films, lower viscosity during processing, low solidification shrinkage of injection moldings (thermotropic processing), and low thermal expansion coefficients. However, the compressive strength of the solid is disappointing. Previous efforts to improve this property have focussed on synthesizing stiffer molecules. The effect of microstructural scale has been overlooked, even though its relevance to the mechanical and physical properties of more traditional materials is well established. By analogy with the behavior of metals and ceramics, one would expect a fine microstructure (i..e. a high density of orientational defects) to be desirable.Also, because much microstructural detail in liquid crystalline polymers occurs on a scale close to the wavelength of light, light is scattered on passing through these materials.

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