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SEMEN and TESTICULAR TISSUE
CRYOPRESERVATION
José Antonio Castilla Alcalá
U. reproducción. H. U. Virgen de las Nieves.
CEIFER. Banco de semen. Granada
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Topics
Legal framework and Guidelines
Sperm Cryodamage
How to optimize semen cryopreservation
Testicular tissue cryopreservation
Biological safety
Special and Future issues
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Guidelines
2010
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Cryopreservation and sperm damage
Cryopreservation is associated with irreversible
structural and functional sperm damage impacting
the recovery of motile and
morphologically normal cells
and the ensuing pregnancy rates
(Nijs and Ombelet., Björdahl et al., 2010)
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Cryopreservation and sperm damage
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0
5
10
15
20
25
30
35
40
% c
ryo
su
rviv
al
All cancer
patients
Prevasect. Cancer
normozoosp
What influences the cryosurvival rate is the quality of the initial
sample, not the fact of having cancer (Agarwal et al., 1995; Hallak et al., 1999; Castilla et al., 2003)
.
Are cancer patients more sensitive to sperm damage in freezing?
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Link between the original sperm quality and their cryosurvival
The lower the initial quality, the lower the cyosurvival rate. •Epididymal Sperm (Holden et al., 1997)
•Ejaculated Sperm (Nijs et al., 2000)
A high percentage (30-70%) of oncological patients
have low quality semen (Lass et al., 1998; Ragni et al., 2003; Bahadur et., 2005;Williams, 2009)
It is very important to optimize
sperm cryopreservation in oncological patients
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¿ How to optimize semen cryopreservation ?
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Biological factor
High variablility between- and within- patients
When possible freezing more than one sample per patient
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We must not delay the start of oncotherapy in order to
increase the period of sexual abstinence
0
5
10
15
20
25
30
35
40
% c
ryo
su
rviv
al
24-48 h 48-72 h >72 h
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Technical factors to optimize sperm cryopreservation
1. Processing semen prior to freezing?
2. Cryopreservation medium
3. Addition/removal medium
4. Cooling rate
5. Freezing system
6. Packaging
7. Storage
8. Thawing rate
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In favor of Density Gradients before freezing
GRADIENTS
BEFORE
GRADIENTS
AFTER
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There is disagreement between authors
GRADIENTS
BEFORE
GRADIENTES
AFTER
In favor of Density Gradients after freezing
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Similar results when different CPMs without
Egg Yolk are compared
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How to optimize sperm cryopreservation?
with Egg yolk Human serum Albumin
Total Semen
Hallak et al., 2000
Zavos et al., 1998
Controversial, animal origin
Proccessed semen
Larson et al., 1997
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Addition/removal of CPM
Drop-wise addition, with continual mixing for several minutes when adding
CPM (freezing)
Culture media (thawing).
Rapid dilution can severely damage cryopreserved spermatozoa
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Straw made from
polyvinyl chloride (PVC),
polyethylene terephthalate glycol (PETG)
Ionomeric resin (more uniform and better
temperature distribution and high biological safety)
Cryovials
Ampoules
(Mortimer, 2004)
Packaging system
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Characteristics of an optimal packaging system
Leakproof
Bacteria- and virus- proof
Mechanically resistant at -196ºC.
2010
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Cryovials take longer in cooling and warming
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Cryovials take longer in cooling and warming
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Freezing system
•Programmable freezing systems
•Static vapor phase cooling
•Mechanically assisted vapor phase cooling
•Flash-freeze technique (ultra-rapid freezing)
•Vitrification
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T ºC
0 9 18 27
20
-196
-10ºC
Time (min)
_
_
_
_
_
Curve Tº semen freezing curve NICOOL LM 10
-120ºC
20 min
5,5 ºC/min
6 min
5ºC/min
Mechanically assisted vapor phase cooling
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Programmable freezing systems
When straws are being used, decrease temperature until <-
132ºC to avoid phenomenon of recrystallization because
they are very sensitive to temperature changes.
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Tomlinson, 2009
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Although some authors have observed lower variability using Controlled freezing
Lowest
variability
(Seeding)
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TYPES OF STORAGE SYSTEM
•Liquid phase nitrogen,
•Vapor phase nitrogen,
•Super cold air
•Mechanical freezers.
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Tomlinson and Sakkas, 2000
No differences have been
observed in pregancy rate per
cycle
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Cooling rate
Slow cooling: 0,1 – 4ºC / min
Fast cooling: 5 – 400ºC / min (Most used)
Ultrarapid cooling: 2500ºC / min
Vitrification: >20000ºC / min
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Cooling rate and survival of different types of cells
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Thawing rate depends on cooling rate
Warmed Room
Temperature (slow) better for slow cooling rate
Warmed 37ºC (rapid) better for
fast cooling rate
Most used
Cooling rate
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The faster the cooling rate,
the faster the thawing rate
3 min
Baño
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Which is the best method for processing cryopreserved semen after thawing?
Density gradients
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Testicular tissue Cryopreservation
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Options for Testicular tissue cryopreservation:
depending on presence of spermatozoa in the sample
Spermatozoa are present
How to cryopreserve?
Cell suspension
Options for fertility restoration
ICSI
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Spermatozoa are present in testicular biopsy
Preparation (Disaggregation)
Mechanical (Scissors, needles, glass slides,..)
With isolation of most dilated tubules
using stereoscope (Kamal et al., 2004)
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Spermatozoa are present in testicular biopsy
Preparation (Disaggregation)
Mechanical (Scissors, needles, glass slides,..)
Enzymatic
Collagenase I o IV
Hyaluronidase II + tripsina
Lysing red blood cell
“Motility-enhancing” chemicals (pentoxifylline)
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Spermatozoa are present in testicular biopsy
Recommendation to freeze without delay/incubation
Increase of DNA fragmentation by 4 hour incubation
Comet assay
(Dalzell et al. 2004)
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Spermatozoa are present in testicular biopsy
Cryopreservation method
Use CPM with Glycerol and Human serum Albumin
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Spermatozoa are not present
How to cryopreserve? Cell suspension
Tissue pieces
Whole testis (need to be developed)
Options for fertility restoration IVM up to a stage at which they are competent for ICSI
Transplantation of purified Cell suspension back to original
testes
Autografting of testicular pieces or whole testis
Xenografting
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Cell suspension
when spermatozoa are NOT present in testicular biopsy
(Experimental technique)
Whether it is better to produce cell suspensions
before or after cryopreservation is still a matter of
debate
Preparation (Disaggregation)
Mechanical
Enzymatic
(Brook et al., 2001) Collagenase I o IV
Hyaluronidase II + tripsina
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Cell suspension
when spermatozoa are NOT present in testicular biopsy
(Experimental technique)
SSC enrichment (2 SSC/
10,000 germ cell)
Techniques
Magnetic-activated cell
sorting (MACS)
Fluorescence activated
cell sorting (FACS)
Markers: PLZF, GFR-α1, Thy-1
SSC
Spermatogonia A
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ANTIGEN SSC
Thy-1 + α6- integrin + CD24 + C-kit - Sca-1 - CD34 - MHC-I - CD9 + Side Population
(BRCP1) -
Phenotype Spermatogonial stem cell
(mouse)
Kubota et al 2003; Lasalle et al 2004; Kanatsu et al 2004
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Cell suspension
when spermatozoa NOT present in testicular biopsy
SSC expansion
Glial-cell derived neurotrophic factor (GDNF)
bFGF
Purification of SSC (detection of cancer cell contamination)
Suboptimal results
Wyns et al., 2010
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Cell suspension
when spermatozoa NOT present in testicular biopsy
No differences
Significant
differences
Brook et al., 2001
CPM
Cooling
rate
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Testicular tissue pieces cryopreservation when spermatozoa NOT present in testicular biopsy
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Wyns et al., 2010
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Vitrification Vitrification medium
DMSO (2.8 mol/L), Ethylene glycol (2.8 mol/L)
25 mg/mL HSA, Diluted in MEM/Glutamax I (Invitrogen)
Three dehydration steps on ice
25% 5 min, 50% 10 min, 100% 10 min
CBS hemi-straw 0.3 mL directly plunged in LN
Warming 37ºC Sucrose (1 mol/L) in MEM/Glutamax I (Invitrogen)
Three decrease step of sucrose
Testicular tissue
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Special issues
How to freeze very very few spermatozoa
Vitrification
Human, mice or hamster zonae pellucida [Cohen
et al., 1997],
Cryoloops [Desai et al., 2004]
Alginic acid beads polymerized by calcium
[Herleer et al., 2006)
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Special issues
If necessary (valuable samples), semen can be
refrozen
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Semen should be refrozen in their original cryoprotectant and not washed
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Use High security straw
All patients should be screened for major viral
markers in advance to minimize the risk of potentially
infective material.
Use different storage tank:
Quarantine tank: for patients without time for screening, and
awaiting the results of the virology laboratory.
Contagious viral disease sperm tank
Emergency or clean tank
BIOLOGICAL SAFETY
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Disinfection tank
Bielanski and Vatja, 2009
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BIOLOGICAL SAFETY
Filling straw using funnel or automatically to
avoid semen coming into contact with the
outside part of straw
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Cleaning the straw after filling and after
thawing before cutting
Using a safe sealing method (Thermal sealing)
BIOLOGICAL SAFETY
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Sterilization of liquid nitrogen with ultraviolet
irradiation (Parmegiani et al., 2010)
BIOLOGICAL SAFETY
Number 4:
With UV
irradiation
Number 3:
Without UV
irradiation
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Conclusions We must not delay the start of oncotherapy in order to increase the
period of sexual abstinence
When possible freezing more than one sample per patient
It is neccesary to optimize technical factors of ejaculated and testicular sperm cryopreservation
Use High security straw
Glycerol, fast cooling and warming rate for sperm
Testituclar tissue pieces (experimental technique)
DMSO and controlled slow cooling
We have to take into account aspects of biological safety in semen and testicular tissue cryopreservation
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Thank you for your attention and your hospitality. I hope to return your hospitality, next September in
Sevilla