The search for excellence2

The search for excellence inthe IVF laboratory

Giles Palmer-Senior Embryologist

Mitera IVF

Factors Affecting IVF Outcome

History, Protocol, Stimulation Endometrial Receptivity

Embryo Transfer

Equipment Staff Laboratory Set up

Techniques Offered

Culture Media Culture ConditionsAir Quality

Flexibility Future Developments

IVF Laboratory

Regulations

• EU Directive 2004/23/EC: Setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells-

-to be implemented in all member countries

• Greek law 2008: ΠΔ 26/2008 εναρμόνιση προς την οδηγίας 2004/23/ΕΚ

• Greek National Authority:Terms and Conditions of licensing Assisted Reproduction Units

-awaiting final approval

Air quality effect on embryos

• Ambient air and its potential effects on conception in vivo

Cohen Hum. Repro 12.8. (1997)

Scientific Publications

• Revised Guidelines for Human Embryology and Andrology LaboratoriesFertil.Steril 82.6. (2004)

• Revised Guidelines for good practice in IVF laboratories

Hum. Repro 23.6.(2008)

Air Quality and Regulations

Clean Room

• 45m2-serving 2 theatres• Clean room Class C• HEPA filters• Airlock, secure entry• Positive pressure• Stainless steel furniture,

Corian© walls• Constant temperature• Constant UPS

Clean Room

• Construction with non-volatile chemicals

• Class C clothing• Microbial counts

Culture Conditions• Mimic in vivo conditions• Highly sensitive to changes

pH, temp, osmolarity• Culture media-basic salt

solution?• Embryo culture requires:

gaseous atmosphere,

pH 7.2-7.4, 280-290mOSM/kg

Temperature 37oC

Culture Conditions

Development up to 8 cells

• Low biosynthetic activity

• Low oxygen tension

• Pyruvate –energy source

• Non-essential amino acids

Development after 8 cells• Activation of embryonic genome

• High biosynthetic activity

• Higher oxygen tension

• Glucose as energy source

• Essential and non essential amino acids

Quality Control in the Laboratory

History, Protocol, Stimulation Endometrial Receptivity

Embryo Transfer

Equipment Staff Laboratory Set Up

Techniques Offered

Culture Media Culture ConditionsAir Quality

Flexibility Future Developments

The Cornerstone of an IVF Laboratory is Quality Management

Quality Control

“Write what you do - and do what you write”

• ISO 9001:2000, 17025:2000, 15189:2003• Written protocols-SOP’S (Τυπικές Διαδικασίες

λειτουργίας )• Constant monitoring of products• Constant monitoring of laboratory conditions• Benchmarking-evaluation of performance• Trouble-shooting- dealing with problems

Quality Control in the Laboratory

Temperature control - pH control -Batch control

Quality Control in the Laboratory

• Safety • Benchmarking

Selecting the Best Embryo(s)

First Appearances Count

*

First Appearances Count

Garello. Hum. Reprod. 14.10. (1999)*

Pronuclei Orientation

Pronuclei Orientation

Balaban. Hum. Reprod. 16.11. (2001)

Blastocyst Culture• Improvement of culture media-more

successful extended culture

• Higher implantation rate of blastocysts • Less chromosome abnormalities

• Morphological assessment Expansion and hatching status (1-6 ) Inner cell mass score (A,B,C )

Quality Trophectoderm (A,B,C )

• Different strategies Day 5 Only Flexible Day 2-3 or 5 What ever the approach- it relies on

morphology-difficult to predict

51% Day 5 embryos preselected on day 3 -Rijenders. Hum. Reprod. 13.8. (1998 )

1AB

2BB

4BB

4AA

Complete Scoring System• Pronuclear morphology• Timing of 1st division• Cleavage patterns• Presence of vacuoles, multi-nuclei• Number of cells/stage

Invasive Techniques

• Vrettou, Palmer et. Prenat. Diag.19. (1999)

• Palmer at al. Hum. Repro.17.1.(2002)

• Embryo selection via embryo biopsy and DNA analysis

• Mitera/Dept. Medical Genetics. Athens University Medical School. St. Sophia's Children's Hospital

Pre-implantation Genetic Diagnosis of Beta Thalasseamia

• 2009 Wide range of Genetic disorders

• Still recommend Prenatal Diagnosis

Invasive Techniques Embryo selection, Improvement of

IVF outcome - PGS???

• Aneuploidy screening-Reduce miscarraige rate, higher pregnancy rate

• Effectiveness remains to be established Staessen, Hum. Repro 13.1.(2007). Twist,

2006. Cochrane review

• Recommendations International Federation of Fertility Societies –

Beneficially only.. 1. ..to improve outcome women > 37-years 2. At least 8 good embryos / 2-3 normal

embryos 3. Highly skilled embryologist 4. 8 preferably 12 chromosomes tested

PCR-monogenic disorders/ molecular level

FISH-ChromosomalAbnormalities

CGH-All chromosomesdetected

Freezing TechniquesProblems with Cryopreservation • Low Temperature – Denaturation of

Proteins• Direct- Intra cellular ice formation• Indirect-Change in ionic interactions

• ‘• hgh

Slow Freezing

• Low concentrations of Cryo-protectant• Cooling rates -0.3oC/min• Controlled ice formation

Vitrification• High concentrations of Cryo-protectant• Cooling rates -2000 to 20000oC/min

Freezing Techniques• Quick method

• Favoured for Oocyte and blastocyst freezing

• Better results for oocyte freezing??

• Many studies showing benefits

• It appears that vitrification is superior to slow freezing regarding post thaw embryo survival

Loutradi Fertil Steril (2008)

Process involves solidifying liquidswithout crystallization to form a gel-like State

The Near Future

Old Techniques improved, Experience gained

• Non invasive assessment of embryo

• Better selection of sperm

• Better culture conditions

Metabolomics

• Systematic study of the unique chemical fingerprint that in vitro embryo leave behind

• Leese & Conaghan- late 1980’s- pyruvate uptake and embryo viability

Leese & Conaghan- Bioessays 15 ( 1993 )

Metabolomics• Embryo Quality does not always relate

to embryo viability and pregnancy

• Sakkas- biochemical factors representative of embryos that give pregnancy or no pregnancy

• Bio-spectroscopy- metabolic profile/ with bioinformatic analysis =Viability scores

• Predictability 80%

• Non invasive, Quick Advantage over other “Omics”

Proteomic, Transciptomic

• 2009-Automative system of embryo metabolisms

• Sakkas. Fertil Steril 90.6.(2008 )

IMSI/MSOME-Super icsi!• Sperm effect on embryo quality often

overlooked

• Although shortage of cytoplasm-

has 3000 mRNA coding for early embryo development

• ICSI method of choice-good enough?

• MSOME selection of spermatozoa

• Previous failure to implant & severe

teratozoospermia

IMSI/MSOME-Super icsi!• ICSI 200-400X vs. IMSI 6000X

• Study of Sperm nucleus morphology

• Bartoov(2003) pregnancy rate 66% vs. 30%

• Suggests ICSI associated Pregnancy rate is affected by subtle morphological malformations

• Spermatozoa with abnormal nucleus have lower fertility potential

Microfluidics …technology of handling small

volumes of liquids in microscopic environments.

• Polystyrene dishes & tubes different to in vivo condition

• Surplus of media

• Microfluidic devices make it possible to mimic in vivo environment

Microfluidics

• Lower concentration are required to fertilize oocytes

• Improved development of embryos

• Cabrera L, 2006 Improved blastocyst development with microfluidics” Fertil Steril;87:S43

Why beneficial?

• Mimic flow of media in vivo

• Secretion of beneficial factors at close proximity

• Removal of harmful toxins

• Reduction in localised oxygen tension

Embryologists

Εμβρυολόγοι του ΜΗΤΕΡΑ IVF

Κουρή Aγγιολίνα

Mαζοκοπάκη Kατερίνα

Μποτζάκη Μητροδώρα

Παπανίκου Χαρά

Σιαλακούμα Aφροδίτη