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 ???