QC in the ART

Laboratory

Ph.D. Lars Johansson, Clinical & Scientific Advisor,

Director of Embryology,

ljohansson@origio.com

+46 709 925460

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• Basic Requirements

• Environment

• Quality Management

• Quality of Service

• Optimization of Outcome

AIM

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Basic Considerations

•Allocate Rooms & Functions • Evaluations vs Treatments?

• Building Materials, Furniture & Equipment’s

• HVAC (constant temp, particles, VOCs)

• Gas Support (Pure CO2, mix & N2)

• Liquid Nitrogen

• UPS – Outlets (Height & Numbers)

• Transport & Deliveries – Easy Access

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Seasonal Variations in

VOCs, TQE & CPR

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Contaminations Affecting Outcome

•Components form Outside Ambient Air

•Laboratory Recirculated Air

•Tissue Culture Ware

•Equipment Off-Gassing

•HVAC Specific Organisms

•Gas Cylinders - contaminations

•Personnel Bioburden

VOCs MOST HARMFUL!!

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VOCs vary in their polarity, molecular

weight and biochemical structure

Require different mechanisms of removal.

Must target all biochemical families.

Increase the effectiveness.

Resonance(removal) and dwell(hold) time.

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Analysis of TVOC Loading & CPR

within the IVF Laboratory

Cli

nic

al P

reg

na

nc

y R

ate

(%

)

Testing Quarters (TQ)

2.7 2.7

2.2 2.2

16.0% 16.0%

37.5% 37.5% 33.3% 33.3%

41.6% 41.6%

13.3% 13.3%

29.4% 29.4%

41.6%

2.7

2.2

16.0%

37.5% 33.3%

41.6%

13.3%

29.4%

0.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5 TV

OC

Le

ve

ls (

pp

b)

51.6%

TQ12 0

10

20

30

40

50

60

70

80

90

100

TQ11 TQ10 TQ9 TQ8 TQ7 TQ6 TQ5 TQ4 TQ3 TQ2 TQ1

54.5% 50.0% 52.4%

0.1 0.1 0.1 0.1 0.1

64.7%

55.5%

The presence of 2.2 ppb VOCs

severely compromised clinical outcomes

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Third Party Independent Testing:

2 CODA Towers + HEPA Final Filters

Source Air IVF

Laboratory

TVOC 1324 ppb 1372 ppb

Biologicals:

Spores 469 cfu/m3 1778 cfu/m3

Particulate

0.3 um 2,318,663 11,642

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LIFE-Aire VOC Free Ventilation System

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LIFE-Aire VOC Free Ventilation System

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Cleanliness: Tightness Factor

- Clean Air – In/Outlets - Exchange: >20/h

- Over-Pressure - Lab: 30 PA

- Pre-room: 15 PA

- Pharmacy Hatches &

Pressurized Door

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•Quality Manager & Quality Manual

•Covers ALL Aspects

•Clinic (Financial, Facility, Safety, Patient Care, Staff etc)

•Treatments Offered

•Team Work - All Staff Involved & Committed

Total Quality Management (TQM)

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Quality Management

•QC: Quality Control • Provide routine and consistent checks to ensure

performance meets predetermined set criteria

• Identify and address errors and neglected items

• Documented and archived (paper & wireless)

•QA: Quality Assurance • Verify that quality criteria has been met.

• Financial, management and service satisfaction.

• Key Performance Indicators (KPI)

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Alarm!

Audible /

visual

alarming

SMS

alarming

Data visualization / management via LAN / web interface

relay

output

mains

24 hours Equipment Surveillance Network (Planer - T, CO2, pH, Humidity, Gas, Particle Count)

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•Routine, robust and reliable • Laboratory, but not limited to the laboratory

•Facilitate Auditing

•Benchmarking against other programs

•Exchange Ideas / Studies

•Customer Satisfaction

QA & International KPI

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Laboratory KPI – Timely Evaluations

David Mortimer, ALPHA 2012

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QA & Factors

•Factors affecting Outcome & Interpretation

•Facility & Laboratory Function

•Patients

• Demographics & Selection

• Batch Treatments

•Expertise / skills (Education, New Staff)

•Time of evaluation (Suitable Intervals)

•Products (Hormones, Disposables, Media)

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Control Charts for Different

Parameters (KPI) • Plot mean of variables each month (KPI)

• Average of monthly means expected value

• Benchmark value

• Control limits = s.d

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QA charts

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70

70

80

90

100

90

92

94

96

98

100

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month 0

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15

20

But use prospectively…Log Book

40

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60

70

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Quality Improvements (QI) – Continuously Performed

FOCUS +

The Devil is in the Details

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Education & EQA in Folliculometry,

Andrology & Embryology

•Samples or via Internet

•SIRT, www.eqasrm.com.au

•AAB, www.americanmedicalacademy.org

•FertAid – www.fertaid.com, QAPonline

•Swiss Clinical Embryologists

•UK NEQAS/ACE EQA

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Total Quality Management (TQM)

•Risk Management – Adverse Events

• Complaint Handling

• Analyse (Root Cause), identify, eliminate & avoid

• Management System

• Describe Processes & Procedures

• Flow Charts – Process Mapping

• Standard Operational Procedures (SOP)

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Standard Operational Procedures (SOPS)

•Written Instructions

•Increase in Standardization

•Efficiency

•Transparency

•Traceability

• Controlled Activities

• Reduce Complaints & Failures

•Facilitates Trouble-Shooting (Auditing)

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Total Quality Management Manual

• Organisational Hierarchy

• Clinics Mission, Vision & Quality Statement

• Staffing

• Position within the Organization

• Job Description

• Duties, Responsibilities

• Education & Competency (Written Plan)

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Pro-Active Actions (SOPs)

1) Cleaning of Premesis and Equipment’s

2) Dress Code / Behavior

3) Hand Hygene

4)Preparation of dishes

5)Selection of disposables

6) Waste Handling

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• Specific for Human ART?

• Certified Quality?

• CE marked (93/42/EEC)

• FDA 510k

• TGA

• Quality Tested with several tests

• MEA

• Sperm survival

• Endotoxin (LAL)

Media Products & Disposables - QC

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VOC Levels: Dishes

0,0

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6,0

7,0

VO

C (

pp

m)

VOC Levels in Culture Dishes

NUNC

Petri Dish

#50255

(0.7 ppm)

NUNC

4-Well Dish

#144444

(1.4 ppm)

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0,0

1,0

2,0

3,0

4,0

5,0

6,0

7,0

8,0

9,0

VO

C (

pp

m)

VOC Levels in Culture Tubes

BD FALCON

5ml Tube

#352058

(6.8 ppm)

VOC Levels: Tubes

NUNC

11ml MEA

Tube

#137860

(0.6 ppm)

NUNC

1.8 ml

CryoTube

#177280

(1.2 ppm)

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Eliminate MisIdentification –

Adverse Effects

• Double Identification

• Labelling of dishes

• Barcodes

• Stickers (www.fertgms.com)

• Laser Etching (www.ankdatasystems.com)

• Electronic Witnessing

• Increase Traceability

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QC - Standard Semen Analysis

• Concentration • Total number of sperm per ejaculate

• Motility • Deletions within mitochondrial DNA is associated with

reduced motility (Ozmen et al., 2007)

• Morphology “THE FORM OF THE SPERM

HEAD IS MORE IMPORTANT

THAN ITS DIMENSIONS”

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Sperm DNA damage with age

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DNA Packing, IR & CPR

Group Implantation Rate CPR/ET

Per ET Per Cycle

CMA3<44%

43 40 32

44<CMA3<60% 35 33 28

CMA3>60% 29 21 18

Esterhuizen et al, 2000

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Preparation of Sperm &

DNA Fragmentation

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GM-CSF, Sperm Activation & Time

• Ejaculated Sperm (Zambrano et al., 2001)

• Tail Region & Acrosome of Ejaculated Sperm (Naz.,2006)

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Reduce the Effects of the Paternal

Genome on Embryo Development

•Objective Selection of Competent Sperm

•Less Fragmentation (D3)

•Higher Blastocyst Conversion Rate

• Improved Blastocyst Quality

•Higher CPR

•Lower Miscarriage Rate

•IVF!!!!!

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N= 537 patients

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N= 537 patients

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• Anasthesia

• Stimulation Protocols • Dysmorphism – short & high doses

• Temperature Control • Tubal Length – Needle to test-tube

• Needle size (gauge): Bleeding & pain

• Single Lumen

• Test-Tube Warmer (36.5-36.9)

• Speed & Handling

• Pump Pressure

• Calibration & Service

Egg Collection - QC

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•Timing of Scoring Important

•What can/Should we score? • Oocyte Quality

• Aggregation or Clustering of SER • Imprinting Disorders?

• High Biochem & Miscarriage Rates (Montag)

• Large Polar Body (Aneuploidy)

Evaluation & Scoring (Istanbul Consensus, 2011)

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Class II Cabinet or LAF

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QC - Handling of COCs

•Media for rinsing of the COC

• AA, ”goodies”

•Control of temp, pH, osmolarity & oxygen

• Meiotic spindle, embryo development & IR

•Do not cut COCs

• Destroy TZP

•Transfer to Mini-Incubator

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Incubators – Recovery,CO2, pH,O2&Temp.

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DAY 0/2-3 and 2/3-5/6

UTM/GMCSF ?

DAY 0 - 5/6

Fertilization w/wo GMCSF

Cleavage w/wo GMCSF

Blastocyst w/wo GMCSF

SAGE 1Step

Medium

2PN-Blast

5 Days

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57

The ORIGIO® Sequential Series™ = 3 media

TRANSFER

ORIGIO® Sequential

Cleav™

ORIGIO® Sequential

Fert™

ORIGIO® Sequential

Blast™

For the fertilisation of oocytes.

For the culture of 2PN embryos to the 2-8

cell-stage (up to day 3).

For the culture of 4-8 cell-stage embryos to

the blastocyst stage (from day 2/3).

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Formulation Focus Areas

• Robustness - Use of pharmacopeia graded products

• Revised components:

- Optimised ionic composition

- Optimised energy substrate composition

- Optimised amino acid concentrations

- Addition of vitamins to Fert™ and Cleav™

- New glutamine source (L-alanyl-glutamine)

• Introduced components: Hyaluronic Acid

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Control New Seq.

Fertilization Rate (%) 76 73

Cleavage Rate (%) 88 87

Good quality embryos (%) 54 49

Blastocyst Rate (Day 5, %) 53 51

Good quality blastocysts (%) 49 51

CPR per patient (%) 48 62

New Sequential Media

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Sage Single-Step Medium

• Same Results as the Competition

• Culture Techniques

- No need for changing media

- Culture from 2PN – Blastocyst (Day 5)

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Precipitates

Precipitates of Calcium Carbonate occurs when

•Vials/flasks are frequently opened which increases the pH – Use small vials

•If you store aliquots of media under 5% CO2 at low temperature (2-8C).

•Do not aliquot!!!

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Recommended pH of Commercial Media

Swain RBM Online 2010 21:6-16

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% 8 cell embryos

7.1

7.15

7.2

7.25

7.3

N= 96, 431 embryos

Courtesy of Kathy Miller

Optimum pH for Cleavage Medium 7.18-7.25

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N= 39, 567 embryos

Courtesy of Kathy Miller

Optimum pH for Blastocyst Medium 7.28-7.35

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Reducing the negative effects on/off oil

• Mineral oil is a mixture of various hydrocarbons.

• Unsaturated hydrocarbons are more susceptible to

peroxidation (POVs), which is embryo-toxic.

• Light & UV light increase the peroxidation.

• Store in dark.

• Heat and prolonged storage increases peroxidation.

• Store at low temperatures (2-8°C).

• Companies pre-wash oil with water or medium

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Low Oxygen (5-6%) = Low ROS

• Reduce chromosomal

mosaicism:

- Blastomeres

- Sex Chromosomes

• Cleavage Pattern

• Blastocysts • Higher Cell Numbers

• Lower Apoptosis

• On time development

Bean et al., 2002, Dunn et al., 2012

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Other considerations QC Micromanipulation

Which ICSI? Heating, Anti-Vibration, Location.

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QC Equipment’s – Particles, Temp, CO2, O2, pH, VOCs

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ICSI-CUMULASE® • Recombinant

human enzyme (rHuPH20)

• Extremely high purity

> 99% pure

NON-CUMULASE® • All are testicular enzymes

from slaughterhouse animals

• Purity not ensured

many unknown substances

Contents of typical products

Bovine

(Type IV-S)

Ovine

(Hyase)

Human

(Cumulase)

? ? Cumulase

(rHuPH20)

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Regulation of pHi during Oogenesis,

Embryogenesis & Denudation

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70 - 90 min

40- 60 min

Spindle Bridge

MI > MII

MII

Montag 2012 ALPHA

Spindle Dynamics and ICSI

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•Open vs Micro-drops?

• Small drops (20 – 40 ul)

• Airflow & High temperature

• Higher osmolarity

• Bad Embryo Development (Swain et al., 2012)

•Single vs Group Culture • Sequential Evaluation

• Paracrine & autocrine & pH?

• Day 1-3, Better Outcome (Ebner et al., 2010)

• After Compaction, single (Lane, FSA 2008)

Culture Techniques

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Direct cleavage (DC) from 1 to 3

Rubio et al 2012, Fertil & Steril

Cleavage from 1-blastomere to 3-blastomeres in less than 5 hrs

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Blastomere Size and Genetic Health

photomicrographs courtesy of

Dr S Pickering

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De-Selection Cleavage Patterns

t2 t3 t4 t5

cc1 cc2

cc3

t2 ≈ t3 < 5 hrs t4 ≈ t5 ≈ t6

cc1

cc2 cc3

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Aneuploidy / Morphokinetics: PGD

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Factors affecting

morphokinetics…

t2 t3 t4 t5 t8

cc1 cc2 cc3

s2 s3

Age

ICSI Media

pH Oxygen

Aneuploidy Viability?

Etiology

Temperature

Handling

Embryo Development

Stimulation

Maturity

Biopsy

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Day 3 vs Day 5 Biopsy

Age

Group

D3 IR D5 IR D3 AR D5AR

30 – 34 51.6 85.3 50.5 41.2

35 – 39 38.1 63.5 65.1 48.1

40 - 42 31.2 76.3 77.5 60.5

Harton et al., ASRM 2011

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•Electronic witnessing

ID Monitoring – Risk Reduction

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Lasers:

- AHA on Day 3/4/5

Octax

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Assisted Hatching with Laser

• Hands not removed from manipulators.

• Laser fired with foot pedal

• Easier to target cell junctions

• Allows aspiration during ablation

• Movable and no calibration

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Uterine Transfer Medium (UTM) / GMCSF

Effect of hyaluronan-enriched transfer medium Urman et al 2008

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10

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30

40

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60

Clinical pregnancy Implantation

Hyaluronan

Conventional

“…beneficial effect was more

prominent in women >35 years

of age, and in women who had

poor-quality embryos”

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Vitrification - QC

Toxicity of components reduced by

• Speed of handling

• Short exposure to final vitrification solution

• Rapid warming

Survival Rates & Developmental Competency

• Selection of high quality gametes & embryos

• Whole vitrification process optimized

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FET better than fresh?

Kato et al., 2012

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Thank You!

Questions?