SEMEN and TESTICULAR TISSUE

CRYOPRESERVATION

José Antonio Castilla Alcalá

U. reproducción. H. U. Virgen de las Nieves.

CEIFER. Banco de semen. Granada

Topics

Legal framework and Guidelines

Sperm Cryodamage

How to optimize semen cryopreservation

Testicular tissue cryopreservation

Biological safety

Special and Future issues

Guidelines

2010

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)

Cryopreservation and sperm damage

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% c

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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?

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

¿ How to optimize semen cryopreservation ?

Biological factor

High variablility between- and within- patients

When possible freezing more than one sample per patient

We must not delay the start of oncotherapy in order to

increase the period of sexual abstinence

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35

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% c

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su

rviv

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24-48 h 48-72 h >72 h

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

In favor of Density Gradients before freezing

GRADIENTS

BEFORE

GRADIENTS

AFTER

There is disagreement between authors

GRADIENTS

BEFORE

GRADIENTES

AFTER

In favor of Density Gradients after freezing

Similar results when different CPMs without

Egg Yolk are compared

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

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

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

Characteristics of an optimal packaging system

Leakproof

Bacteria- and virus- proof

Mechanically resistant at -196ºC.

2010

Cryovials take longer in cooling and warming

Cryovials take longer in cooling and warming

Freezing system

•Programmable freezing systems

•Static vapor phase cooling

•Mechanically assisted vapor phase cooling

•Flash-freeze technique (ultra-rapid freezing)

•Vitrification

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

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.

Tomlinson, 2009

Although some authors have observed lower variability using Controlled freezing

Lowest

variability

(Seeding)

TYPES OF STORAGE SYSTEM

•Liquid phase nitrogen,

•Vapor phase nitrogen,

•Super cold air

•Mechanical freezers.

Tomlinson and Sakkas, 2000

No differences have been

observed in pregancy rate per

cycle

Cooling rate

Slow cooling: 0,1 – 4ºC / min

Fast cooling: 5 – 400ºC / min (Most used)

Ultrarapid cooling: 2500ºC / min

Vitrification: >20000ºC / min

Cooling rate and survival of different types of cells

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

The faster the cooling rate,

the faster the thawing rate

3 min

Baño

Which is the best method for processing cryopreserved semen after thawing?

Density gradients

Testicular tissue Cryopreservation

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

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)

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)

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)

Spermatozoa are present in testicular biopsy

Cryopreservation method

Use CPM with Glycerol and Human serum Albumin

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

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

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

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

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

Cell suspension

when spermatozoa NOT present in testicular biopsy

No differences

Significant

differences

Brook et al., 2001

CPM

Cooling

rate

Testicular tissue pieces cryopreservation when spermatozoa NOT present in testicular biopsy

Wyns et al., 2010

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

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)

Special issues

If necessary (valuable samples), semen can be

refrozen

Semen should be refrozen in their original cryoprotectant and not washed

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

Disinfection tank

Bielanski and Vatja, 2009

BIOLOGICAL SAFETY

Filling straw using funnel or automatically to

avoid semen coming into contact with the

outside part of straw

Cleaning the straw after filling and after

thawing before cutting

Using a safe sealing method (Thermal sealing)

BIOLOGICAL SAFETY

Sterilization of liquid nitrogen with ultraviolet

irradiation (Parmegiani et al., 2010)

BIOLOGICAL SAFETY

Number 4:

With UV

irradiation

Number 3:

Without UV

irradiation

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

Thank you for your attention and your hospitality. I hope to return your hospitality, next September in

Sevilla