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In vitro culture in the presence of amorphous calcium
carbonate improves mice embryos compaction and
hatching rates
Amir Arav DVM PhD
Consulting to Amorphical LTD
Examples of CaCO3 occurring in nature: mollusk shells
10µm
calcite vaterite
aragonite
Amorphous Calcium Carbonate
ACC seeds
0.1µm
Polymorphism CaCO3
Calcium carbonate nano-particles have a large number of
applications due to their beneficial properties such as high
porosity, high surface area to volume ratio, non-toxicity and
biocompatibility
Amorphous Calcium Carbonate (ACC)
CaCO3 2HCl Ca CO2 2H2O + + +
CaCO3 will only increase pH in acidic environments
Buffering capacity
ACC Control
Compaction D3 (60h)
Control ACC
Dose dependence of ACC
ACC 17µl/ml ACC 34µl/ml
D5 Hatching blastocysts (110h) - 10-20% more hatching
ACC Control
Cleavage medium (Quinn-SAGE) with ACC or CCC
Arav ESHRE 2017
Arav ESHRE 2017
100% hatching blastocysts in the presence of ACC
Testing SAGE one-step medium
Mouse
#
No. of Embryos % Compaction (60h) % Blastocysts (86h) % F. Hatching
without
ACC With ACC
without
ACC With ACC
without
ACC With ACC
without
ACC With ACC
1 13 13 75 80 75 80 50 60
2 19 19 30 67 30 45 13 100
3 18 16 40 89 80 67 70 78
4 13 14 46 86 30 57 61 100
5 14 11 36 90 18 90 27 90
6 10 20 27 70 55 85 0 35
7 14 22 75 100 37 100 37 100
8 20 10 40 67 20 80 20 80
9 9 11 33 100 25 100 25 100
10 10 15 70 90 80 90 80 100
11 15 19 67 78 67 56 78 100
12 19 18 40 60 20 60 30 70
Testing IRVINE CMC medium
Mouse #
No of Embryos % Compaction (60h) % Blastocysts (86h) % F. Hatching
without
ACC With ACC
without
ACC With ACC
without
ACC With ACC
without
ACC With ACC
1 13 11 69 78 54 55 46 60
2 14 14 57 72 43 72 55 72
3 18 20 69 70 54 71 46 100
4 14 14 0 22 21 22 29 100
5 23 29 73 74 20 28 52 62
6 13 15 20 62 31 87 23 80
7 8 11 12 55 0 34 0 37
8 13 11 69 78 54 55 46 57
9 14 13 36 62 43 38 43 70
10 14 13 36 38 43 48 43 92
11 11 15 55 60 36 27 27 60
12 11 14 55 64 36 36 27 68
Arav ESHRE 2017
Cathepsin- Lysosomal Proteases (A,B,D, K,V…) Most of the members become activated at low pH
Richard Willstätter 1872 –1942 Nobel price 1915
Willstätter & Bamann (1929) Hoppe-Seylers Z. Physiol. Chemie 180, 127-143
Intracellular Cathepsin B Activity Is Inversely Correlated With
the Quality and Developmental Competence of Bovine
Pre-implantation Embryos
A.Z. Balboula, K. Yamanaka, M. Sakatani, A.O. Hegab, S.M.
Zaabel AND M. Takahashi
E-Cadherins (named for "calcium-dependent adhesion") are a class of type-1 transmembrane proteins. They play important roles in cell adhesion, forming adherent junctions to bind cells within tissues together. They are dependent on calcium (Ca2+) ions to function.
E-cadherin as a target substrate of cathepsins B, L, S but not C and S
Amorphous CaCO3
Proposed mechanism
Low pH
Ca++ CO3--
Increase pH Reduce Cathepsin activity
Support embryo compaction
E-cadherin
Arav et al., 2017
Low Glucose and low cathepsin
High Glucose and high cathepsin Oxidative Glycolysis
pH 7.6
pH 6.9
Amorphous calcium carbonate (ACC) at a concentration
of 1.7mM acts as a buffer increasing the compaction,
blastocyst and hatching rates of mice embryos.
Conclusion
Yosi ben
Orit Ecker Cohen
Pliensky Ylena
Yehudit Natan
Otto Warburg 1883-1970 Nobel price 1931
Warburg effect
Science, New Series, Vol. 123, No. 3191. (Feb. 24, 1956), pp. 309-314
Proliferation Migration Invasion
Angiogenesis
Aerobic Glycolysis
H --- pHe (6.5-6.9)
Lysosome Cathepsin
Embryo implantation metabolism
E-Cadherine
Metaloproteinase Glycolysis
Oxidative
Arav et al., 2017
H --- pHe (6.5-6.9)
VEGF
+
Lysosome Cathepsin
Metalloproteinase (MMPS)
Cancer cell
Angiogenesis
Acid mediates invasion
Immune response
APOPTOSIS AND NECROSIS
Normal Tissue
Arav et al., 2017
oncogenic
mutations
P53
Out of 8 transfer with >80 embryos none has produced pregnancies
Blastocyst transfer with ACC
