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