Transcript Preservation of germ cells in ART program

Preservation of
Germ Cells in
ART Program
Byung-Rok Do, Ph.D.
Infertility Research Center, MizMedi Hospital
Contents
 I.
Introduction
 II. Principle and Summing up
of Preservation
 III. Applications of
Cryopreservation
 IV. Conclusions
 V. Further Research
I. Introduction
Utility of Preservation
 Banking
/ Preservation
– Safe keeping or storage of utilities for
emergency use
 Cells,
tissues, organs
 Prevention of endangered species
 Genome resource banking
Methods of
Preservation
 Room
temperature
 Subzero
preservation
Methods of
Preservation
 According to objects
– Organ, Tissue, Cells
 Duration of storage
– Short term : Cells, Semen, Sperm
– Long term (subzero) : Cells, Semen,
embryos, oocytes, tissues, ….
Method of
Cryopreservation
Slow
freezing method
Rapid
freezing method
– Ultra rapid freezing
– Vitrification
Aims of
Presentation
 Overview
of the current
technologies and approaches
utilized in preservation
 Consider
the factors affect to
results of cryopreservation
II. Principle and
Summing up of
Preservation
Basic Principle of
Cryopreservation
 In
natural
– Many plants in the winter
– Some plants live in polar region
can recover after cooled below 80℃
– Others ??
Principle of
Cryopreservation
 Water mediate all chemical
reactions in the cell
 Some cells can be preserved for
several months at -80 ℃
– Decrease or prevent of physical and
chemical reactions in the cell
 All
chemical reactions are prevent
under 130 ℃
– Usually preservation under LN2 (-196 ℃)
Factors Affecting
Cryopreservation
 Water
– Crystallization and Volume
increase
– Physical damage to membranes
and micro-organelles in the cell
– Cell death
Factors Affecting
Cryopreservation
 Cryoprotectant
– Membrane permeability
– High osmolarity
– Non-crystallized material
– Easily super cooling
– low melting point
– ethanediol(EG), propanediol(PROH),
dimethyl sulfoxide(DMSO), glycerol,
di-, and triethylene glycol etc.
Factors Affecting
Cryopreservation

Seeding temperature
Buffer
Salt concentration
Non-permeable additives



–

Sugar, raffinose, polyethylene glycol (PEG),
polyvinyl pyrroridone (PVP), Ficoll etc.
Cooling rate
–
–
Materials, species specific manner
Chilling sensitivity
Composition of
Freezing Solution
 Cryoprotectant
– ethanediol(EG), propanediol(PROH),
dimethyl sulfoxide(DMSO), glycerol,
di-, and triethylene glycol etc.
 Non-permeable additives
– Sugar, raffinose etc.
– polyethylene glycol (PEG), polyvinyl
pyrroridone (PVP), Ficoll etc.
Procedure of
Slow Freezing
 Dehydration
– Repair time of volume is correlated with
membrane permeability of cryoprotectant
 Equilibration
 Cooling
until -6 or -7 ℃
 Seeding
– Extracellular ice formation
– Freezing solution must be frozen under
seeding temperature
Temperature (°C)
Procedure of
Slow Freezing
Unfrozen cell
0
Outflow of intercellular water
Extracellular ice
-80 Unshrunken cell
With internal ice
Vitrified intact cell
Time
Shrunken cell
with little or
no internal ice
Cryodamage
 Types
of damage
– Physical
Rapid dehydration and hydration
 Temperature falling
 Shrinkage

– Chemical

Cryoprotectant
Cryodamage
 Cryoprotectant
(1)
– Biphasic
Binding with water and lipid
– Structural changes of lipid bilayer
 Join with membrane lipids
– Polarization or removal of
cytoplasmic lipids

Cryodamage
 Cryoprotectant
(2)
– Raises free tubulin concentration
Spindle size reduced
 Microtubule disorganization

– Chromosome dispersed 10-30 min at room
temperature
Microfilaments disrupt
 Inducing chromosomal abnormality
– Detrimental effect on the ER, Golgi body,
mitochondria, cytosol

Cryodamage
 Temperature
(cold shock)
– Changing lipid membrane permeability
 Cause of Ice crystal formation
 Unrepairable microtuble damage
– Latent heat at Seeding point
Increase temperature
 Salt concentration increase

– Increase outer part osmolarity
– Rapid remove of water

Rapid shrinkage
Cryodamage
 Salts
– Salts concentration relatively increase
– Protein precipitation by salts
– Membrane protein damage including
membrane enzyme
– Membrane function change
– Sodium chloride
 Choline chloride, EGTA, PEG
Cryodamage
 Membrane
– Osmotic shock
 Microorganells
– Dehydration
 DNA
– Ionizing radiation
 Zona-pellucida,
– Blabbing
membrane
Cryodamage
 Increase
reactive oxygen
species
– Mitochondrial damage
– Decreased ATP synthesis
 Cortical reaction
– Zona-hardening
Table. Factors associated with cooling and
cryopreservation that contribute to cellular
injury and death in biological system
System
Type / cause of damage
All
Intracellular ice formation, extracellular ice formation,
apoptosis, toxicity, calcium imbalance, free radicals,
ATP levels, general metabolism, fertilization failure,
cleavage failure, pH, parthenogenesis activation,
cleavage
Membrane
Rupture, leakage, fusion, microvilli, phase transition
Chromosomes
Loss/gain, polyspermy, polygyny (failure to extrude
polar body), tetrapolidy
DNA
Cytoskeleton
Proteins/enzymes
Ultrastructure
Apoptosis, fusion, rearrangements
Microtubles dissolve, actin
Dehydration, loss of function
Microvilli, mitochondria, vesicles, cortical granules,
zona pellucida
Hardening, fracture
Free radicals ?
Zona Pellucida
Lipids
Rapid Freezing
Using high concentration of
cryoprotectant (about 40%)
 Ultra-rapid freezing
– Ice crystal formation
 Vitrification
Rapid Freezing
 Vitrification
– Using cryoprotectant mixture (ca. 40%)
low MW
 high membrane permeability
– High concentration of non-permeable
additives
– Using EM greed, capillary, wire loops,
microdrop, foil

Rapid Freezing
 Advantages
–
–
–
–
–
–
–
–
of vitrification
No ice crystal formation
Rapid equilibrium
Absence of Water leak after equilibrium
Decreased osmotic shock
Shot time required
Minimum damage of membrane lipid
Simple procedure
No equipment require
Rapid Freezing
 Disadvantages
of vitrification
– Protocol establishment is required
– Ultra rapid thawing is required
Ice crystal formation during
hesitating thawing procedure
– Test for genetic damage need to
carry out

III. Applications of
Cryopreservation
Applications of
Cryopreservation
 Cells,
tissues, organs preservation
 Prevention of endangered species
 Genome resource banking
 In ART
– Oocyte, embryo, semen, ovarian tissue,
testis tissue preservation
– Patients for chemo- or radio-theraphy
1. Embryo
Applications

–
–
–
–
–
For maximum pregnancy achieve in a
single stimulation
Decrease the risk of multiple
pregnancy
Reduce the risk of OHSS
Synchronization for donation program
Other no ET patient
1. Embryo
 Trounson and Mohr, 1983
– Glycerol and DMSO, Slow freezing
 Methods
– Slow freezing
– Rapid freezing
– Ultra rapid freezing
– Vitrification
1. Embryo
 Optimal time of preservation (1)
– PN stage
 General stage for cryopreservation
– 20-22 hours after fertilization
– PN scoring method
Freezing media ; PROH, sucrose
 Established method
 Higher pregnancy rate than cleaved
stage

1. Embryo
 Optimal
time of preservation(2)
– Cleaved stage

Easy conform for embryo quality
1. Embryo
 Optimal
time of preservation(3)
– Blastocyst stage
Limitation the number of transferred
embryo
 Natural selection
 Unknown risk of long term in vitro
culture
 Reduced multiple pregnancy
 Cryoprotectant : glycerol, DMSO

2. Oocyte
 Chen et al., 1986
– DMSO, Slow freezing
 Less
ethical and legal issue then
embryo freezing
 Preservation of ovarian function
– Cancer or POF patients
 Oocyte bank
– for infertility patients
2. Oocyte
 Risk of freezing
– Increase the chance of chromosome
aneuploidy after thawing
 Spindle fiber damage
– Polabody extrusion, movement of PN,
cytokinesis damage
 Microfilament damage
– Zona hardning or damage
 cortical granule exocytosis
2. Oocyte
– Relatively good fertilization and
developmental rate
– Lower viability and pregnancy rate
then embryo
– Various pregnancy rate was
reported according to researcher
– High abortion rate
– Farther research needed
2. Oocyte
 Cryopreservation method
– DMSO, PROH, slow freezing method
– EG, Vitrification
 Rafaella Fabbri, HR, 2001
– PROH, slow freezing method
– High concentration of additives
– Higher viable rate than conventional
method
3. Ovarian tissues
 Primordial
follicles in ovarian
cortex
 Preservation of fertility to the
patients have cancer or POF
 None ethical and legal problem
 Not well established the method
and treated to the patients, yet
3. Ovarian tissues
 Transplantation
– Patients
 Steroid synthesis under adipose
tissue
– Animal
 Antrum formation in SCID mouse
 May
be a good method for
infertility treatment
A
C
B
D
Figure. Microphotographs of 22 weeks old human fetal ovarian slices
and neonatal mouse ovary culture for 3 weeks in vitro.
A: Fresh human fetal ovary, B: Frozen-thawed and 3weeks
cultured human fetal ovary, C: Fresh mouse neonatal ovary,
D: Frownd thawed mouse ovary for 3 weeks culture
A
B
C
D
Figure 1. In situ RT-PCR and immunohistochemical localization for FSH receptor in human ovaries. FSH receptor gene
expression was recognized on 20 weeks old fetal (A) and 3 days old neonatal ovaries (B) using In situ RT-PCR method.
Localization of FSH receptor was identified on 20 week-old fetal (C) and 3 day-old neonatal ovaries (D) using
immunohistchemical method. Thin arrows, germ cell cord; thick arrows, primordial follicle; arrow heads, streamed cell.
Bars present 100 μm.
A
B
D
E
C
F
Figure . Microphotographs of 22 weeks old human fetal ovarian slices. A, histological
section of the frozen-thawed ovaries stained with hematoxylin-eosin. Arrows, germ cell;
Immunohistochemical stainings with proliferating cell nuclear antigen. Arrow, primordial follicle:
B, frozen-thawed control; arrow, primordial follicle; thin arrow, germ cell; C, BSA only group;
arrow, primordial follicle; D, hrFSH 10IU/ml + VIP 10g/ml; arrow, germ cell; E, hrFSH 10IU/ml;
F, VIP 10g/ml; Arrows, germ cell. Fetal ovarian slices were cultured for 48 hours. Bar, 50mm.
A
B
C
D
Figure . Microphotographs of 22 weeks old human fetal ovarian slices immunohistochemically stained with Ki67
antigen. A, frozen-thawing control; B, BSA only; C, hrFSH 10IU/ml; and D, VIP 10g/ml. Arrows, germ cells. Fetal ovarian
slices were cultured for 48 hours. Bar, 50mm.
A
B
C
D
Figure. Microphotographs of FSH receptor gene expressed on 22 weeks old human fetal ovarian slices detected by in
situ PCR. A, H-E stain; B, thawing control; C, negative in situ PCR; and D, hrFSH 10 IU/ml + VIP 10mg/ml. Ovarian slices
were cultured for 48 hours and conducted in situ PCR to detect the expression of FSH receptor genes, Arrows, primordial
follicles; thin arrows, FSH receptor gene detected by in situ PCR. Bar, 25mm. Original magnification, X1,000.
1
2
3
4
5
6
7
8
9
838bp
600bp
359bp
Figure. Semiquatitative RT-PCR of FSH receptor and b-actin genes. Lane 1, 100bp ladder marker; 2, positive
control using immature follicular granulosa cells; 3, thawing control; 4, BSA only; 5, VIP 10mg/ml; 6, VIP
100mg/ml; 7, FSH 10IU/ml; 8, FSH 10IU/ml+VIP 10mg/ml; 9, FSH 10IU/ml+VIP 100mg/ml. Base pairs (bp) of
RT-PCR products of B-actin gene and FSHR gene were 838bp and 359bp, respectively.
3. Ovarian tissues
 Problems at present
– Optimizing in vito culture conditions of
primordial follicles
 maturation, fertilization, and
development
 Methods
of ovarian tissue
cyopreservation
– PROH, Slow freezing
– No different results between
slow freezing and vitrification
4. Sperm and
tseticular tissue
 Applications
– Man with a possibility to lose the
fertility by cancer or other diseases
– Low counts of sperm
– Asynchronous time of ejaculation
and ovulation in the process of IUI
or IVF
4. Sperm and
tseticular tissue

In non-obstructive azoospermia, the
testicular tissue is obtained by the
biopsy, and then cryopreserved these
tissue is used in ART

Cryoprotectants
– Glycerol, DMSO
– Additives ; increase the permeability of
sperm or cell membrane
 + ions, milk, egg yolk, fructose, citrate
4. Sperm and
tseticular tissue
 Cryopreservation methods
– Sperm: rapid freezing by the vapor of LN2
– Testicular tissue: programmed slow
freezing
 Thawing
– Sperm survival rate: effect of rapid change
of osmolarity by the freezing solution
– Cell membrane damage by cryoprotectant
IV. Conclusion
IV. Conclusion
 Embryo,
sperm, testicular
tissues are generally use and
achieve by frizen-thowed
programs in Human ART
program
 Can
use the frozen thawed
oocyte and ovarian tissues
IV. Conclusion
 General
week point of
cryopreservation
– Complex
– High priced equipment
– long time spent
– Safety
V. Further Research
 Simplify
– Vitrification
 Safety
 Animal
Model
– Ant, Bee
– Hen
New
method ???