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Embryo Viability Morphology and Mitochondria

• Determining embryo viability
• Morphological assessment
• Bilateral symmetry and the polar body
• Mitochondria

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Embryo Viability
How do we know which embryos are good?

• When to assess?- oocyte, zygote, 4-8-cell,
  blastocyst
• How to assess?- biopsy-polar body, blastomere,
  morphology
• What scoring system to use?- Cumulative Embryo
  Score (CES), Embryo Quality (EQ), Embryo
  Development Rating (EDR), ...

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Oocyte Scoring

• Expansion and radiance of cumulus cells
• Meiotic status- MII
• Polar body morphology
• Refractile bodies, vacuoles
• Darkened or unhealthy cytoplasm
• Fractured zona pellucida, large perivitelline
  space

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Zygote Scoring
Nucleolar precursor bodies does not differ
gt3 polarization with lt7 in each not
lt3 polarization in both
Good
Bad
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Embryo Scoring
EQ Symmetry/Size of blastomeres, fragments in
perivitelline space, quality of cytoplasm EDR
Ratio between time of embryo at a stage and time
expected to be at stage
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CES
CES(grade)(
blast.)1(grade)( blast.)2
Grading 1. Extensive fragmentation and morph.
aberration 2. Moderate fragmentation, gt1 morph.
aberration 3. 1 morph. aberration or lt3
blastomeres 4. gt2 equal blastomeres, smooth
memb., clear cytoplasm
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The Polar Body and Bilateral Symmetry

• At least one polar body (PB) remains present to
  blastocyst stage
• PB attached by weak tether, doesnt move
• PB aligned with animal-vegetal axis
• PB aligned with greater diameter in blastocyst

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PB
PB
Here is the trick. The text didnt match the
picture exactly. They rotated it 90?, but
through both axes not just one.
74.9 ?m
81.0 ?m
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Mitochondria in the Oocyte and Embryo

• Use products of glycolysis to produce energy-
  ATP
• Located in areas of energy demand
• Associated with microfilaments and
  microtubules?
• Defective or low numbers of mitochondria
  correlated with reduced developmental capacity
• Mouse oocyte contains 92,500 mitochondria
  (1.19x105 or 1.59x105 copies mtDNA). Human
  oocyte contains 3.14x105 copies of mtDNA.

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Where are the Mitochondria???
PERINUCLEAR!!

• Many species studied mouse (5), hamster, human
  (3)
• Rhodamine (Rh123), nonyl acridine orange(NAO)
• Oocyte- homogeneous?perinuclear
• Early embryonic stages- perinuclear, follows
  cell cycle
• Exception nonblocking embryos aggregated in
  cytoplasm
• Compaction- in the cortex?
• Blastocyst- perinuclear in trophectoderm

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Nucleus
Zona pellucida
Homogeneous
Aggregation
Perinuclear
Cortical
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Where do we go from here?

• Mitotracker on oocytes, embryos
• Mitotracker IS NOT Rhodamine 123, it is used
  after fixing cells
• Confocal microscopy
• Correlate QTM to look for mitochondria

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Key References
Barnett DK, Kimura J, and Bavister BD. (1996)
Translocation of active mitochondria during
hamster preimplantation embryo development
studies by confocal laser scanning microscopy.
Developmental Dynamics 205 64-72. Fukada A,
Breuel KF, and Thatcher SS. (1993) Localization
and denxity of mitochondria in the mouse embryos
developed in vivo and in vitro. Human
Reproduction 128 47. Tokura T, Noda Y, Goto Y,
and Mori T. (1993) Sequential observation of
mitochondrial distribution in mouse oocytes and
embryos. Journal of Assisted Reproduction and
Genetics 10(6) 417-426. Muggleton-Harris AL, and
Brown JJG. (1988) Cytoplasmic factors influence
mitochondrial reorganization and resumption of
cleavage during culture of early mouse embryos.
Human Reproduction 3(8) 1020-1028. Batten BE,
Albertini DF, and DucibellaT. (1987) Patterns
of organelle distribution in mouse embryos during
preimplantation development. The American
Journal of Anatomy 178 204-213. Neganova I,
Sekirina GG, and Eichenlaub-Ritter U. (2000)
Surface-expressed E-cadherin, and mitochondrial
and microtubule distribution in rescue of mouse
embryos from 2-cell block by aggregation.
Molecular Human Reproduction 6(5) 454-464. Noto
V, Campo R, Roziers P, Swinnen K, Vercruyssen M,
and Gordts S. (1993) Mitochondrial distribution
after fast embryo freezing. Human Reproduction
8(12) 2115-2118. Sathananthan AH and Trounson
AO. (2000) Mitochondrial morphology during
preimplantational human embryogenesis. Human
Reproduction 15(Suppl. 2) 148-159. VanBlerkom J,
Davis P, and Alexander S. (2000) Differential
mitochondrial distribution in human pronuclear
embryos leads to disproportionate inheritance
between blastomeres relationship to microtubular
organization, ATP content and competence. Human
Reproduction 15(12) 2621-2633. Bavister BD and
Squirrell JM. (2000) Mitochondrial distribution
and function in oocytes and early embryos. Human
Reproduction 15(Suppl. 2) 189-198.