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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
3
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
4
• 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
5
• 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
10
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
11
• 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
15
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%
16
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
17
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
18
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
19
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
20
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
* *
22
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
23
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
24
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
25
Vitrification Automation – Genea Experience
Hum Reprod. 2014 Nov;29(11):2431-8
26
Automated Equilibration
Roy et al., 2014. Hum Reprod. Nov;29(11):2431-8
Roy et al., 2014. Hum Reprod. Nov;29(11):2431-8
27
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!