Tammie Roy Genea Biomedx Sydney, Australia. Declared to be stakeholder in Genea Biomedx

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1 Tammie Roy Genea Biomedx Sydney, Australia Declared to be stakeholder in Genea Biomedx 1

2 24-25 September 2015 Madrid and Alicante, Spain Importance of cryopreservation in Assisted Reproductive Technology IMPROVING THE PATIENT S LIFE THROUGH MEDICAL EDUCATION

Cryopreservation in ART Sperm Cryopreservation Preservation of

counts Testicular and epididymal sperm Oocyte Cryopreservation

sample unavailable Low responder Embryo Cryopreservation Excess

3 Cryopreservation in ART Sperm Cryopreservation Preservation of fertility (cancer) Donor programs Partner unavailable Low sperm counts Testicular and epididymal sperm 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 3

4 Cryopreservation Challenges 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 4

5 Vitrification or Slow Freezing 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 5

6 6 Slow Freezing

7 Slow Freezing Freeze Control Planer BioCool Biotronics 7

8 8 Vitrification

9 9 Vitrification Devices

10 Vitrification Devices Open or Closed Open Devices (e.g. Cryotop) Faster Cooling/Warming rates Direct exposure to LN 2 Closed Devices (e.g. HSV) Slower Cooling and/or Warming rates No direct contact with LN 2 10

11 Sperm Cryopreservation 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 11

12 12 Cryopreservation of single (or few) sperm

13 13 Oocyte Vitrification

14 14 Oocyte Vitrification

15 Blastocyst Vitrification Genea Experience Survival 83% Imp. Rate 25.2% 2010-now Pre 2006 CVM Fibreplug TM Slow Freezing Survival 81% Imp. Rate 21.4% Kitazato Cryotop 15

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: M 05: M Up to 15:00 01: ml 01: M 03:00 0.5M 16

2006- Dec 2007 1002 Fibreplug TM Cycles Mar 2010- Feb 2012 1247 Kitazato Cryotop cycles Analysed:

17 Comparison of two vitrification methods Fresh Vitrified Retrospective analysis Fresh group had at least one blastocyst vitrified Jan Dec cycles Mar Feb cycles Jan Dec Fibreplug TM Cycles Mar Feb Kitazato Cryotop cycles Analysed: - For all groups: - Ave Age - Implantation Rate - For vitrified groups: - Recovery - Survival 17

Implantation Rates for Fresh Blastocysts 42.5% 35.7 44.8% 35.7 49% 34.0 53% 33.4 40.0 31% 32% 39.

18 Implantation Rates for Fresh Blastocysts 42.5% % % % % 32% % 50 Average Age % 25 0% 0 n = TOTAL <38 >=38 18

19 Recovery and Survival Rates 83.4% 98% 94.1% 99.7% 84.3% 97.6% 93.5% 99.6% 83.6% 95% 95.6% 100% Recovery Survival 100% p<0.001 * * p<0.05 p<0.001 * * p<0.05 p<0.001 * * Device 1 Device 2 50% 0% n = Total <38 >= 38 19

20 Implantation Rates 25.2% % % % % 26% 40.5 Device 1 100% 50 p<0.001 p<0.05 Average Age p<0.001 p<0.01 Device 2 * * * * * * 50% 25 0% 0 n = TOTAL <38 >=38 20

21 Fresh or Frozen Roy et al., Fertil. Steril, 101:

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 22

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 23

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 24

25 Vitrification Automation Genea Experience Hum Reprod Nov;29(11):

26 Automated Equilibration Roy et al., Hum Reprod. Nov;29(11): Roy et al., Hum Reprod. Nov;29(11):

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

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! 27

EGG BANKS program at clinique ovo

EGG BANKS program at clinique ovo HISTORY The first pregnancy resulting from egg donation in an IVF cycle was in 1983 Egg donation plays an important role in assisted reproductive technologies since it