Preservation of Preservation of Germ Cells in Germ Cells in ART Program ART Program Byung-Rok Do, Ph.D. Byung-Rok Do, Ph.D. Infertility Research Center, Infertility Research Center, MizMedi Hospital MizMedi Hospital
Jan 15, 2016
Preservation of Preservation of Germ Cells in Germ Cells in ART ProgramART Program
Byung-Rok Do, Ph.D.Byung-Rok Do, Ph.D.Infertility Research Center, MizMedi HospitalInfertility Research Center, MizMedi Hospital
ContentsContentsI. IntroductionI. IntroductionII. Principle and Summing up II. Principle and Summing up
of Preservationof PreservationIII. Applications ofIII. Applications of
CryopreservationCryopreservationIV. ConclusionsIV. ConclusionsV. Further ResearchV. Further Research
I. I. IntroductionIntroduction
Utility of Utility of PreservationPreservation
Banking / PreservationBanking / Preservation– Safe keeping or storage of utilities for
emergency use
Cells, tissues, organs Prevention of endangered speciesGenome resource banking
Methods of Methods of PreservationPreservation
Room temperature
Subzero preservation
Methods of Methods of PreservationPreservation
According to objects– Organ, Tissue, Cells
Duration of storage– Short term : Cells, Semen, Sperm– Long term (subzero) : Cells, Semen,
embryos, oocytes, tissues, ….
Method of Method of CryopreservationCryopreservation
Slow freezing method
Rapid freezing method–Ultra rapid freezing–Vitrification
Aims of Aims of PresentationPresentation
Overview of the current technologies and approaches utilized in preservation
Consider the factors affect to results of cryopreservation
II. Principle and II. Principle and Summing up of Summing up of
Preservation 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 CryopreservationWater mediate all chemical
reactions in the cellSome 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), dim
ethyl 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 Composition of Freezing SolutionFreezing SolutionCryoprotectantCryoprotectant
– ethanediol(EG), propanediol(PROH), dimethyl sulfoxide(DMSO), glycerol, di-, and triethylene glycol etc.
Non-permeable additivesNon-permeable additives– Sugar, raffinose etc. – polyethylene glycol (PEG), polyvinyl pyrr
oridone (PVP), Ficoll etc.
Procedure of Slow Freezing
Dehydration – Repair time of volume is correlated with mem
brane permeability of cryoprotectant
Equilibration Cooling until -6 or -7 ℃Seeding
– Extracellular ice formation– Freezing solution must be frozen under seedi
ng temperature
Procedure of Slow Freezing
Tem
pera
ture
(°
C)
0
-80
Unfrozen cell
Outflow of intercellular water
Extracellular iceShrunken cell with little or
no internal ice
Unshrunken cell With internal ice
TimeVitrified intact cell
CryodamageTypes of damage
– PhysicalRapid dehydration and hydrationTemperature fallingShrinkage
– ChemicalCryoprotectant
CryodamageCryoprotectant (1)
– BiphasicBinding with water and lipid
– Structural changes of lipid bilayerJoin with membrane lipids
– Polarization or removal of cytoplasmic lipids
CryodamageCryoprotectant (2)
– Raises free tubulin concentrationSpindle size reducedMicrotubule disorganization
– Chromosome dispersed 10-30 min at room temperature
Microfilaments disruptInducing chromosomal abnormality
– Detrimental effect on the ER, Golgi body, mitochondria, cytosol
CryodamageTemperature (cold shock)
– Changing lipid membrane permeability Cause of Ice crystal formation Unrepairable microtuble damage
– Latent heat at Seeding pointIncrease temperatureSalt concentration increase
– Increase outer part osmolarity– Rapid remove of water
Rapid shrinkage
CryodamageSalts
– Salts concentration relatively increase– Protein precipitation by salts– Membrane protein damage including me
mbrane enzyme – Membrane function change
– Sodium chloride Choline chloride, EGTA, PEG
CryodamageMembrane
– Osmotic shock
Microorganells– Dehydration
DNA– Ionizing radiation
Zona-pellucida, membrane – Blabbing
CryodamageIncrease reactive oxygen s
pecies – 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
Zona Pellucida
Lipids
Apoptosis, fusion, rearrangements
Microtubles dissolve, actin
Dehydration, loss of function
Microvilli, mitochondria, vesicles, cortical granules, zona pellucida
Hardening, fracture
Free radicals ?
Rapid Freezing
Using high concentration of cryoprotectant (about 40%)
Ultra-rapid freezing– Ice crystal formation
Vitrification
Rapid FreezingVitrification
– Using cryoprotectant mixture (ca. 40%)low MW high membrane permeability
– High concentration of non-permeable additives
– Using EM greed, capillary, wire loops, microdrop, foil
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
Rapid FreezingDisadvantages 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 ofIII. Applications of Cryopreservation Cryopreservation
Applications of CryopresApplications of CryopreservationervationCells, tissues, organs preservationPrevention of endangered speciesGenome 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, sucroseEstablished method Higher pregnancy rate than cleaved stag
e
1. Embryo
Optimal time of preservation(2)
– Cleaved stageEasy conform for embryo quality
1. EmbryoOptimal time of preservation(3)
– Blastocyst stageLimitation the number of transferred embr
yoNatural selectionUnknown risk of long term in vitro cultureReduced multiple pregnancy Cryoprotectant : glycerol, DMSO
2. OocyteChen 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. OocyteRisk of freezing
– Increase the chance of chromosome aneuploidy after thawing
Spindle fiber damage – Polabody extrusion, movement of PN, c
ytokinesis damageMicrofilament damage
– Zona hardning or damagecortical granule exocytosis
2. Oocyte– Relatively good fertilization and de
velopmental rate – Lower viability and pregnancy rate
then embryo– Various pregnancy rate was report
ed according to researcher– High abortion rate – Farther research needed
2. OocyteCryopreservation 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 meth
od
3. Ovarian tissuesPrimordial follicles in ovarian
cortexPreservation of fertility to the
patients have cancer or POFNone ethical and legal problem Not well established the method
and treated to the patients, yet
3. Ovarian tissuesTransplantation
– Patients Steroid synthesis under adipose tissu
e– Animal
Antrum formation in SCID mouse
May be a good method for infertility treatment
A C
B DFigure. 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 10g/ml; arrow, germ cell; E, hrFSH 10IU/ml; F, VIP 10g/ml; Arrows, germ cell. Fetal ovarian slices were cultured for 48 hours. Bar, 50mm.
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 10g/ml. Arrows, germ cells. Fetal ovarian slices were cultured for 48 hours. Bar, 50mm.
A
C D
B
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.
A B
C D
1 2 4 5 6 7 8 93
359bp
838bp600bp
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.
Problems at present– Optimizing in vito culture conditions of pr
imordial folliclesmaturation, fertilization, and developm
entMethods of ovarian tissue cyoprese
rvation– PROH, Slow freezing – No different results between slo
w freezing and vitrification
3. Ovarian tissues
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
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 spe
rm 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
4. Sperm and tseticular tissue
IV. Conclusion
IV. ConclusionEmbryo, sperm, testicular tiss
ues are generally use and achieve by frizen-thowed programs in Human ART program
Can use the frozen thawed oocyte and ovarian tissues
IV. ConclusionGeneral week point of cry
opreservation
–Complex –High priced equipment– long time spent–Safety
V. Further V. Further ResearchResearch
Simplify–Vitrification
SafetyAnimal Model
–Ant, Bee–Hen
New method ???