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Tammie Roy Genea Biomedx Sydney, Australia

Declared to be stakeholder in Genea Biomedx

www.excemed.org

IMPROVING THE PATIENT’S LIFE THROUGH

MEDICAL EDUCATION

Importance of cryopreservation in Assisted

Reproductive Technology

24-25 September 2015

Madrid and Alicante, Spain

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Cryopreservation in ART

Oocyte Cryopreservation

• Preservation of fertility (cancer, age)

• Donor programs

• OHSS

• Semen sample unavailable

• Low responder

Embryo Cryopreservation

• Excess embryo production

• OHSS

• Better outcomes

• Poor endometrial preparation

• Cumulative pregnancy

Sperm Cryopreservation

• Preservation of fertility (cancer)

• Donor programs

• Partner unavailable

• Low sperm counts

• Testicular and epididymal sperm

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• Intracellular water represents 80% of cell volume

• Ice crystal formation can destroy cellular membranes

• Early embryos depend on a small number of cells to survive

• Oocyte cryo is even more complex due to the fact that: • Is one big cell with↓ surface-to-volume ratio and ice crystal formation

• Low permeability of oocyte cell membrane causes slower movement of Cryoprotective agents (CPs)

• Meiotic spindle of MII oocyte very sensitive to chilling and can become easily disorganised

Cryopreservation Challenges

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• Some labs around the world are still doing slow-freezing (blastocysts)

• Shift towards vitrification in last 10 years in IVF due to: • Increased survival and pregnancy rate

• Less requirement for equipment

• Reduced time for process

Vitrification or Slow Freezing

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Slow Freezing

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Slow Freezing

Planer BioCool Freeze Control Biotronics

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Vitrification

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Vitrification Devices

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Open Devices (e.g. Cryotop) • Faster Cooling/Warming rates

• Direct exposure to LN2

Closed Devices (e.g. HSV) • Slower Cooling and/or Warming rates

• No direct contact with LN2

Vitrification Devices – Open or Closed

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• First successfully cryopreserved human cells

• Allows for quarantine period for donor samples

• Ejaculate cryopreservation considered easy due to high number of sperm

• Surgically retrieved testicular more difficult due to low count and immotility

• Mostly done with slow freezing using glycerol as the main cryoprotectant (with or without controlled rate freezer)

• For surgically retrieved samples: • Important to remove large tissue

• Can lyse the red blood cells

• Concentrate the sample and then dilute with sperm cryopreservation medium

Sperm Cryopreservation

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Cryopreservation of single (or few) sperm

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Oocyte Vitrification

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Oocyte Vitrification

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Blastocyst Vitrification – Genea Experience

Slow Freezing

CVM FibreplugTM

Kitazato Cryotop®

Pre 2006

2006-2009

2010-now

Survival – 81% Imp. Rate – 21.4%

Survival – 83% Imp. Rate – 25.2%

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Equilibration 8%EG/8% DMSO

Vitrification 16%EG/16% DMSO/ 0.68 M Trehalose

Trehalose Warm Solution 1

Trehalose Warm Solution 2

03:00 00:35 2 ml

05:00 0.33 M

05:00 0.2 M

Up to

15:00 01:30 0.1 ml

01:00 1.0 M

03:00 0.5M

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Comparison of two vitrification methods

Fresh

Jan 2006- Dec 2007 1303 cycles

Mar 2010- Feb 2012 1597 cycles

• Retrospective analysis • Fresh group had at least

one blastocyst vitrified • Analysed:

- For all groups: - Ave Age - Implantation Rate

- For vitrified groups: - Recovery - Survival

Vitrified

Jan 2006- Dec 2007 1002 FibreplugTM

Cycles

Mar 2010- Feb 2012 1247 Kitazato

Cryotop® cycles

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Implantation Rates for Fresh Blastocysts

3

4.0

4

0.0

39

.8

3

3.4

35

.7

35

.7

TOTAL

42

.5%

44

.8%

<38 >=38

49

%

31

%

32

% 53

%

100%

50%

0%

2006-2007

2010-2012 Average Age

50

25

0

n = 1303 1597 940 1009 363 588

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Recovery and Survival Rates

Recovery Survival

Total

98

%

9

9.7

%

<38 >= 38

97

.6%

95

%

10

0%

9

9.6

%

100%

50%

0%

Device 1

Device 2

8

3.4

%

94

.1%

83

.6%

9

5.6

%

8

4.3

%

9

3.5

%

p<0.001

* *

p<0.05

* *

p<0.05

* *

p<0.001

* *

p<0.001

* *

n = 1424 1459 1018 894 406 565

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Implantation Rates

3

3.8

4

0.6

40

.5

3

3.6

35

.7

36

.2

TOTAL

25

.2%

37

.1%

<38 >=38

28

%

18

% 26

%

44

%

100%

50%

0%

Device 1

Device 2

Average Age 50

25

0

n = 1002 1247 723 771 279 476

p<0.001

* *

p<0.001

* *

p<0.01

* *

p<0.05

* *

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Fresh or Frozen

Roy et al., 2014. Fertil. Steril, 101:1294-1301.

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Freeze All

• Advantages: • Better outcomes?

• Increased cumulative pregnancy

• Preparation of endometrium

• Reduced influence of stimulation regime on endometrium

• Disadvantages: • Requirement for very good cryopreservation technique

• Patient understanding

• Time and cost

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Overview of Current Vitrification

Manual Vitrification

• Advantages:

• Fast freezing and warming rates • Excellent embryo survival • Increased pregnancy rate compared to slow freezing • Quicker than slow freezing??

• Disadvantages: • Steep learning curve • Training can be lengthy • Time consuming with high volumes • Issues with open versus closed systems

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To Automate or Not to Automate?

• Advantages: • Automate to improve vitrification consistency

• Standardise protocol to deliver consistent results

• Reduce training requirements

• Eliminate LN2 contamination risk through completely closed system

• Improve lab productivity by better utilising embryologist time

• Disadvantages: • Cost

• Reliance on equipment

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Vitrification Automation – Genea Experience

Hum Reprod. 2014 Nov;29(11):2431-8

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Automated Equilibration

Roy et al., 2014. Hum Reprod. Nov;29(11):2431-8

Roy et al., 2014. Hum Reprod. Nov;29(11):2431-8

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Conclusions

• Vitrification is the way to go

• Need to control as many variables as possible

• The better we become the more we will freeze and…

…the higher the cumulative pregnancy rates!

• More automation equipment is needed to: • Control the equilibration process in vitrification

• Have repeatability across global laboratories

• Increased laboratory efficiency – time savings

• The future will certainly go in this direction!