Quality management in IVF to optimise embryo transfer results James Catt PhD Adjunct Senior Lecturer Scientific Director Dept of Obs and Gyn Optimal IVF Monash University Melbourne Australia

Quality is about continuously rejecting the status quo and is a journey, not an end

Definitions Quality Assurance Design of a process to deliver the defined product In IVF lab = design of all procedures to optimise the chances of implantation

Definitions Quality Control Examines the product during the process to ensure specifications are met In IVF lab = monitoring individual procedures to meet expectations

Audits Third party monitoring of QA and QC Auditor looks at procedure documentation and results

Summary Quality system - laboratory and unit QA/QC Staff education Document Control Risk Management Audit cycle

Procedure design (QA) Provide optimal conditions Equipment Ambient conditions Media Embryo selection

Procedure monitoring (QC) Ie What quantitative outcomes during the IVF process can be used to monitor the programme ?

Quality management of IVF instrumentation Selection of appropriate equipment Validation Ongoing QC

Equipment selection Availability, reliability, service then cost Central question to ask is ‘what happens if this piece of equipment fails?’ Backup!!

Selection: Should we believe the manufacturers? All equipment comes with specifications. Do they truly reflect what the embryos experience? Eg incubators Where are the temperature probes?

Should we believe the manufacturers? Behind here !

Validation: Should we believe ourselves? Independent equipment eg thermometers, dataloggers, CO2 meters have to have calibration traceable to a standard

Dataloggers Short, medium and longterm fluctuations occur in all equipment Dataloggers measure these fluctuations and therefore help determine suitability of equipment

Center SE 309 4 channels 8 000 datapoints Datalogger Center SE 309 4 channels 8 000 datapoints

Standard Incubator Thermal image

Benchtop Thermal image

Validation: pH How do you measure pH ? XpH meter X Blood gas analyser Colour pH paper!!!!!

Paper into medium Read within 3 seconds How do you use pH paper ? Paper into medium Read within 3 seconds

Validation: Toxicity testing MEA? Sensitized sperm survival assay (low protein or CASA)

EQUIPMENT QC: How often? As much as necessary! Tendency to over audit Eg incubator

Incubator overaudit

Ambient conditions Equipment in, set up and monitored What about laboratory conditions?? Gametes and embryos spent some time outside of incubators

Air purity Temperature pH Ambient conditions Air purity Temperature pH

Volatile Organic Compounds (VOC) Therefore particles per se are not detrimental until they have VOC adsorped onto them. Therefore should only need to monitor for VOC Elimination Filters or photocatalytic destruction

Ppm X Ppb 

Data from clinic 16% FH/embryo ET 100 ppb 48% FH/embryo ET 100 ppb

Temperature Surprising where variation comes from !! Laminar flow Stage warmers Pipetting

Laminar flow Temperature drop

Laminar flow

Stage warmers Stage warmers will not heat a dish for ~5 min !! Petri dish Air = insulation Warm stage

Heat Transfer in Pipettes (1) Liquid (37°C) into pipette (2) Immediately heat is lost to the walls the pipette Lab Air (3) The surface of the pipette loses heat to the surrounding air Line of symmetry 33

What to do ? Partial solution is to use plastic pipettes Other solution is to control environment

Solution to most environmental problems – control the environment!

The forgotten Oocyte

The forgotten Oocyte Good oocytes make good embryos Do NOT use simple media for oocyte recovery Eg saline, PBS etc

Inadequate sperm Sperm have specific requirements High glucose/fructose High protein Bicarbonate

Mixing media DO NOT MIX MEDIA !! All have different components and will affect embryo homeostasis if mixed

Quality control to select the ‘best’ embryos Objective outcomes Consistency Attainable goals

(hours post-insemination) Expected stage of development Type Timing (hours post-insemination) Expected stage of development Fertilization check 17 ± 1 Pronuclear stage Syngamy check 25 ± 1 Expect 50% to be in syngamy (up to 20% may be at the 2-cell stage) Early cleavage check 27 ± 1 2 cell-stage Day 2 embryo assessment 44 ± 1 4-cell stage Day 3 embryo assessment 68 ± 1 8-cell stage Day 4 embryo assessment 92 ± 2 Morula Day 5 embryo assessment 116 ± 2 Blastocyst 41

Laboratory outcomes Average number of oocytes collected* Average number of oocytes suitable for ICSI IVF fertilization rate* ICSI fertilization rate* ICSI degeneration rate* Syngamy rate (25+/- 1 hpi) FH per embryo transferred* Utilisation rates Percent survival of thawed embryos* FH per thawed embryo* FH per transferred thawed embryo* * Denotes suitability for individuals

Human Embryonic development OPU Fertilization syngamy Day 2 ET Day 3 Freeze D5/6 Day 5 Day 4

Consistency Good prognosis group Age most important ‘Good prognosis ’ group should reflect the majority of patients and exclude the ‘difficult’ ones Eg <39 and < 3 previous cycles

How do we measure the quality of oocytes entering our laboratories?? SYNGAMY (or early cleavage)!!

Oocyte quality Early syngamy 40% 25%

Other factors affecting implantation Day of transfer Developmental stage

Results eSET (FH/embryo)

Conclusions Single embryo culture and single embryo transfer has enabled important factors in embryo development to be identified Early cleavage and blastocyst expansion are the best predictors for implantation Implantation rates for eSET can exceed 50%

Cumulative pregnancy rates The total number of fetal hearts from a stimulated cycle when both fresh and frozen embryos have been transferred

Results Day of pregs patients Cumulative Rate fresh ET 3 913 1393 66% 3 913 1393 66% 5 228 356 64% No significant difference

Conclusions There is no decrease in the cumulative pregnancy rate with extended culture There are no ‘extra’ pregnancies with extended culture There are fewer transfers to achieve the pregnancy

QC Summary Know and control your equipment Know and control your conditions Quantitate your outcomes Benchmark your outcomes