2 IVf Lab Setup and QC.pdf

2 IVf Lab Setup and QC

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Welcome
Street No1, AshokStreet No1, AshokViharViharColony, Opp.Colony, Opp.MandigateMandigate,,
PandriPandri, Raipur, Raipur(Chhattisgarh)(Chhattisgarh)
Visit us:Visit us:www.ivfandicsiindia.comwww.ivfandicsiindia.com

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DR. (Mrs.) Ratna Agrawal
Clinical Embryologist (Leeds, UK)
HospitalHospitalDirectorDirector
Ashoka Super Speciality Women HospitalAshoka Super Speciality Women Hospital
& IMSI, ICSI Test Tube Baby Centre, Raipur (C.G.).& IMSI, ICSI Test Tube Baby Centre, Raipur (C.G.).
•Dr. Ratna Agrawal is the Director & the Chief Embryologist of Ashoka Super Speciality
Hospital & IMSI, ICSI Test Tube Baby Centre, Raipur.
•Fellowship in Belgium & Sydney IVF Australia in Specialized Techniques IVF & ICSI.
•She has presented scientific papers in many conferences and she conducted teaching
programme for the Gynaecologist in different IUI Techniques.
•She is the Central Member of ISAR Society.
•She is the General Secretary cum Treasurer of ISAR Chhattisgarh Chapter.
•She is the member of Alpha Scientists in Reproductive Medicine
•She was the Organizing Secretary of 17th ISAR National Congress which was held in
Raipur on 1, 2, 3, & 4th of March, 2012.
•She has attended so many National and International Infertility Conferences.
•She was awarded by Governor of Chhattisgarh State in Women day’s for good social
work.
•Recently she achieved the Clinical Embryology degree from Leeds University UK.

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IVF Laboratory Setup
and Quality Control
By
Dr. RATNA AGRAWAL
Clinical Embryologist
(Leeds University, UK)
Director,Ashoka Superspeciality Women Hospital,
& IMSI, ICSI, Test Tube Baby Centre, Raipur (C.G.).
The various aspects that need
planning
•Layout of the Lab and basic infrastructure
•Personnel
•Laboratory equipment and consumables
•Main areas of the IVF lab
•Overall Management Strategy
•Future Expansions

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LAYOUT OF AN IVF LAB
•Away from traffic to reduce dust and noise
pollution and Vibrations
•Restricted Access to the lab preferably
through air locks
•Next to the operation theatre (Ovum-pick-
up/ Embryo Transfer Room) should be the
culture lab to minimise temperature loss to
oocytes.

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IVF LAB BASIC INFRASTRUCTURE
•ELECTRICITY
•WATER SUPPLY
•AIR
•FLOOR & WALL CEILINGS
•FURNITURE & WORKING PLATFORMS
IVF LAB BASIC INFRASTRUCTURE
Electricity
•Sufficient power points with properearthingand
current ratings should be provided.
•Power points should be at appropriate height and
easily accessible.
•Provision of emergency power supply–UPS &
Generator is must.
Preferably, the main Circuit should be kept outside
the Lab. So that repairs will be provided without
access to lab.

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IVF LAB BASIC INFRASTRUCTURE
WATER SUPPLY
•Water sinks and taps in IVF lab should be avoided.
•Ultra-pure(doubledistilled)Watershouldbe
usedforIncubatorHumidificationandwashingof
labware.
•Scrubbingandwashingareaswillneedadequate
watersupplyandwell-concealeddrainage.
IVF LAB BASIC INFRASTRUCTURE
AIR
•IVF Lab demands cleanest air
•The lab air should be filtered through HEPA
Filters
•Air Pressurizing Apparatus for maintaining
positive pressure inside the lab could be
installed
•Comfortable and constant temperature
should be maintained by split air
conditioning systems with Bio-Filters
•VOCs (Volatile Organic Compounds) and
CACs (Chemical Air Contaminates) in the
lab should be eliminated by using
specialized filters like Coda Towers etc

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IVF LAB BASIC INFRASTRUCTURE
Floors & Walls Ceilings
•The floors should be non-slip, easily cleanable and with
minimum joints
•Thewallscouldbetileduptoceiling
•Thetilesshouldbesuchthattherearefewerjointsand
surfaces,whichmayaccumulate,dustparticles
•POPFalseCeilinginIVFlabisnotadvisableforits
inaccessibilityformaintenanceinfuture
•Modulareasy-toaccessfalseceilingsareavailable
IVF LAB BASIC INFRASTRUCTURE
Furniture and Working Platforms
•The working benches should be of a correct and
comfortable height.
•The surface should be of non-porous and cleanable
material–No wood in the lab.
•Chairs and stools with adjustable height add to the
comfort of the scientists and reduce fatigue
•Easy sliding steel trolleys with drawers with adequate
capacity for storage should be installed in the lab under
the working benches
•It is preferable not to put shelves on the walls as dust
can collect on top of them and go unnoticed

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PERSONNEL
One dedicated program Director/coordinator with-
•Gynaecologistwell conversant with
reproductive medicine
•Ultrasonologist
•Andrologist
•Embryologist
•Councellor
LABORATORY PERSONNEL
•Healthy interaction among clinical and laboratory
personnel have major impact on the success of the
PGM.
•Highly skilled technician should share talents with
other less able persons.
•Downturn situations should be handled carefully.
•Instead of finger-pointing to deflect blame, all
personnel will seat together to identify the cause.
•Outside consultant may be considered.

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LIST OF EQUIPMENTS
•CO
2Incubator (Preferably 2) & working Incubator
(Optional).
•Stereo dissecting Microscope
•Stage warmer
•Centrifuge machine with Digital Recorder.
•Laminar Air Hood (IVF Tech)
•Inverted Microscope with Micromanipulator.
•Bio-freezer with storage tank fitted with alarm.
•Refrigerator
•Osmometer, pH meter, VOC meter
Assisted Laser Hatching / Oocyte Spindle view/
Fluorescence microscope (optional) / Embryoscope
List of Disposables/ Media
•Petridish
•Culture dish-multidish
•1ml pipette
•Conical centrifuge tube
•Round bottom tube.
•Tissue culture flask
•Millipore filter
•Syringe
•Ovum pick-up needle.
•Transfer catheter.
•Sperm buffer / Medium
•Follicle flushing medium
•Fertilization / Cleavage /
blastocyst medium
•IVF media
•Consumables & media
needed for sperm / embryo
cryopreservation
•Consumable & media
needed for thawing sperm /
Embryos.

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MAIN AREAS OF IVF LAB
•Changing Room
•Scrub / Wash Area
•Labware /Media Storage
•Andrology (Semenology) Lab
•Private room for semen collection
•OT for Pick-up & Insemination
•IVF-Culture Lab with Micromanipulator
•Ultrasound room
•Freezing/Cryopreservation Room
•Record room
CHANGING ROOM
■A ward-robe to accommodate
routine clothes of the IVF Staff
with mirror.
■Special Lab Gowns or Overcoats.
■Disposable Face-Mask and Caps.
■Special Lab Slippers a waste-bin
/ Laundry Bag.

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Scrub /Wash Area
•Just before entering Lab/OT
hands should be washed
•Stainless Steel Deep Sink
•Elbow or Foot Operated Tap
Controls and Soap Dispensers
•7X Detergent / 70% Alcohol
•Dryer or Paper Towels
•Dust Bin
Labware / Media Storage
•Adequate clean storage
space for Labware should be
provided near IVF lab
•It should be properly sealed
and pest and termite proof
•Culture Media should be
stored in a dedicated
refrigerator
•A small storage trolleys for
daily requirement in the lab

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Record Maintenance
•Consent of the couple should be maintained thoroughly and
also computerized.
•Important forms for records include:
–Consent forms–especially documenting if the IVF /ICSI is
done with the husband or donor sperm.
–Request forms–where the clinician orders the IVF /ICSI
–Reports forms-pre and post processing parameters, where
the sample produced and who did the processing &
procedure.
–Details documentation for freezing ofgametes & embryos.
–Disposal forms(consent forms) to dispose the stored
gametes & embryos.
Andrology Lab
•Laminar Flow Cabinet (preferably
Class II)
•Mini Incubator
•Warming blocks for swim-up test
•Phase / contrast microscope up
to 400X
•Sperm counting chamber
•37
0
C based Centrifuge machine.
•Refrigerator /Freezer

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SEMINOGRAM
MaklerChamber Phase/Contrast
Microscope up to 400X
Oocyte Aspiration (Pick-Up) Room
•Next To Embryology Lab
connected by a pass-thru
tunnel or hatch
•Ultrasound scanner with
abdominal & transvaginal
probe
•Examination couch and lab
chairs
•Monitor for anesthesia
•Aspiration pump
•Heating Block or Mini
Incubator

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IVF-Culture Lab
Laminar FlowIVF-Culture Lab

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New Trends in Assisted Reproduction
In vitro egg maturation (IVM)
Assisted Laser Hatching
Blastocyst culture
Cytoplasmic transfer
Mild IVF
Preimplantation Genetic Diagnosis(PGD)
Frozen Embryo Transfer (FET) /Cryopreservation
Intra-Cytoplasmic Morphologically Selected Sperm Injection(IMSI)
Embryoscope
Computer Assisted Semen Analysis (CASA)
MICROMANIPULATER WITH INVERTED
MICROSCOPE(JAPAN)
ICSI PROCEDURE

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Intra-Cytoplasmic Morphologically
Selected Sperm Injection (IMSI)
IMSIhelps to select best sperms based onmorphology
to improve the success and is said to be more beneficial
than ICSI in patients with
Two previous IVF or ICSI failures
Unexplained infertility
Severe male factor infertility
Better advantage in terms of higher pregnancy rate and
lower miscarriage rates.
It helps in magnifying the image of the sperm 7,200 times,
thereby allowing to pick the best looking morphologically
healthier sperms.
IMSIVsICSI

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Embryoscope
EmbryoScopeis anovel embryo monitoring systemfor
assessing embryo quality.
UsingLeicaoptics it provides continuous control and
recording of embryo developmentand provides
Respiration ratesof single embryos during development
image acquisition of embryo development
onset and duration of cell divisions
In future Embryoscope may replace the classical
microscope based methods for selecting embryos
EMBRYOSCOPE:EMBRYO MONITORING SYSTEM

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Computer Assisted Semen Analysis(CASA)
•Calculate the spermatic Counts & Concentrations of
a sample
•Detailed results on Motility & Progressive Motility,
velocities, motion characteristic
•Morphology and morphometry
•DNA fragmentation study
•Makes the assessment of semen quality fast,
accurate more subjective and detailed.
Pre Implantation Genetic Diagnosis
PGDis recommended most frequently for :
–Patient with unexplained infertility
–Recurrent miscarriages
–UnsuccessfulIVFcycles
–Advanced maternal age
–Sever male factor infertility
It enables diagnosis of agenetic disorderin an
embryo before its implantation in the uterus.

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Pre Implantation Genetic Diagnosis
BlastocytTransfer
Repeated failureto achieve pregnancy
following the transfer of good quality
cleaved embryos
To achieve pregnancy without therisk of
multiple pregnancy.
Patient who do not wish to have their spare
embryos frozen for whatever reasons may
be advised to have blastocyst transfer.

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Assisted Hatching : Methods
The main methods currently
in use for assisted hatching are:
Chemical
Mechanical
Laser–
allows grater degree of control and precision
Indications for Assisted Hatching
Older Age > 38 yrs
ElevatedFSH
Egg quantity and quality factor
Embryo qualitypoor quality embryos
(excessive fragmentation or slow rates of cell division)
Zonapellucidathickness>17 mm
PreviousIVFfailures
Cryo-preserved Embryos
Embryo generated fromIVM
Helps to increase pregnancy rates by improving
implantation rates.

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Cryopreservation
Cryopreservation has become anintegral component of
assisted reproductive technology.
Cryopreservation techniques are being developed for
gametes, embryos & gonadal tissues.
Freezing of embryos allows significant chance of
pregnancy after single ovarian stimulation
Assuming all embryo survive freeze/thaw process the
patient can undergo 2 cycles of transfer in a typical retrieval
cycle of 15 oocytes
The pregnancy rates of Vitrification technique is promising
Laboratory Equipment
Main Criteria
•Good Quality Control
•Preventive Maintenance programs.

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Total Quality Management
•QualityControlisdesignedtocheckthatapiece
ofequipmentwillproducethesameresultvery
time.
•QualityAssuranceisdesignedtomonitor&
evaluatetheentireprocessinthelab.
•QualityAuditcheckingtheSOPandre-arranging
theprocedureifrequired.
•Guidelines for human embryology andandrology
laboratories by the American Fertility Society
(1992)
•Guidelines for good practice in IVF laboratories
by the European Society for Human
Reproduction and Embryology (ESHRE) (2000)
•Reproductive Laboratory Accreditation
standards, College of American Pathology (2002)
•Accreditation standards and guidelines for IVF
laboratories by the Association of Clinical
Embryologists (2002)

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CO
2-Incubators
Monitoring of Incubators
Temperature
•Record digital display
•Check actual temperature by a calibrated
thermometer
CO2
•Record digital display
•Check actual value by a calibrated instrument
•Monitor CO2 tanks pressure
pH
•Monitor in new batches of culture media
Weekly and Monthly checks
Liquid Nitrogen tanks
•Monitor level using ruler-weekly
Refrigerators and freezers
•Monitor using calibrated thermometer
inside the cabinet-monthly
Heated working surfaces and water baths
•Check tube warmers and warm surfaces
with a calibrated thermometer

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Evaluation End Points
Short term
•Grading of Oocyte maturity
•Fertilization/degeneration rates following ICSI
•Grading embryo quality
•Evaluation of sperm parameters
Long term
•Pregnancy rate / transfer
Indicators to be Assessed are
•Numbers of errors/adverse events
•Rates of top quality embryos
•Proportion of patients with failed fertilization
•Ongoing clinical pregnancy rates (fresh & frozen)
•Multiple pregnancy rates
•Implantation rates
•Rate of survival of embryos after thawing

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Safety
•Personnel
•Gametes
•Patient
Infectious Agents
•Vaccination of staff
•Mandatory screening of patients and donors
•The laboratory staff must be informed about
the risks of handling infected biological
material.

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Safety Concerns
Verification of patients' identity-
–before ovum retrieval
–at semen recovery
–embryo transfer
Double checks must be done:
–Insemination of oocytes
–Replacement of embryos
–Embryo freezing and thawing
Protective Measures
•Strict observation of staff hygiene regulations.
–Food, drinks, cigarettes and cigars are strictly forbidden.
–The use of make-up and strong perfumes should be
limited.
•Use of laboratory clothing.
•Use of non-toxic (non-powdered) gloves and masks
•Use of adequate protection if cryogenic materials are
handled.
•Use of vertical laminar-flow benches.
•Use of mechanical pipetting devices
•Appropriate disposal

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“WHEN THE SOIL IS“WHEN THE SOIL IS
WELL PREPARED,WELL PREPARED,
THE GARDEN WILLTHE GARDEN WILL
GROW”.......GROW”.......

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The Ultimate Goal: Making Better Embryos
Record ofIVF,ICSI&IMSIcases in last 17 years
–5680 cases
IVFSuccess rate20 to 25%
AfterICSI,Success rate35 to 40%
AfterIMSI, Laser Assisted Hatching &Vitrification,
Success rate55 to 60%
Take Home Baby Rate45.66%
AshokaSuperSpecialityWomen Hospital
& IMSI, ICSI TestTube Baby Centre

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Success Graphs
Ashoka Super Speciality Hospital &
IMSI, ICSI Test Tube Baby Centra, Raipur
Conclusion
•A well plannedIVFLaboratory from the initial
setup itself would be conducive to the growth of
the services required and the results obtained.
•New trends in ART haverevolutionisedthe
reproductive medicine in last one decade.
•These new technologies have also improved
success rates in ART.

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Visit us:www.ivfandicsiindia.com
Cell:094255-03700
E-mail:[email protected]