Manual G5 INTL v5-0-5

G-SERIES Manual
Recommended use of G-Series by Gardner

Edition 7, January 2013
20022013 Vitrolife Sweden AB. All rights reserved.

You may copy this for internal use only, not for publishing.
The Vitrolife logotype is a trademark of Vitrolife Sweden AB, registered in Europe, the
U.S. and other countries.
Vitrolife Sweden AB
Box 9080
SE-400 92 Gteborg
Sweden
Tel: +46-31-721 80 00
Vitrolife Inc.
3601 South Inca Street
Englewood
Colorado 80110
USA
Tel: +1-866-VITRO US (866-848-7687)
G5, G-Series, the G5 logo, GIII, GIII Series, the GIII logo, G-MM, HSAsolution, G-IVF, G-IVF PLUS, G-1, G-1 PLUS, G-2, G-2 PLUS,
G-GAMETE, G-MOPS, G-MOPS PLUS, G-RINSE, G-FreezeKit Blast,
G-ThawKit Blast, G-PGD, SpermGrad, HYASE, ICSI, FreezeKit Cleave,
ThawKit Cleave and V-Tip are trademarks of Vitrolife Sweden AB.
EmbryoGlue is a registered trademark of Vitrolife Sweden AB.
Vitrolife G-Series Manual 6.0
Contents
Introduction 4
General 5
Opening your Vitrolife products
5
CO2 equilibration
6
Supplementing media
6
Recommended use of G-MOPS 8

How to measure pH
10
IVF and Culture System
12
Sperm preparation
Swim-up method (migration)
Density gradient centrifugation method
13
14
16
IVF-ET 19
Follicle aspiration
19
Oocyte identification
20
Oocyte culture and fertilisation
21
Insemination 22
Fertilisation assessment
22
Scoring of human pronuclear embryos
23
Choosing the culture system
24
Preparation of culture system
25
Embryo culture
26
Embryo assessment
28
Scoring criteria for human blastocysts
29
Scoring system
30
Table for assessment of blastocysts
31
Blastocyst schedule
32
Embryo transfer
33
Replacement of embryos
33
Loading the catheter
35
Micromanipulation 37
Denudation of oocytes for ICSI
38
ICSI procedure
40
Difficult ICSI cases
42
Testicular biopsy
43
Embryo biopsy
43
Embryo Cryopreservation
45
Equipment list
45
Cryopreservation of cleavage
stage embryos using Freezekit Cleave
and Thawkit Cleave
47
Cryopreservation of cleavage
stage embryos using Freezekit 1
and Thawkit 1
49
Cryopreservation of blastocyst stage
embryos 52
Quality Control Program
56
G-Series and related products

from Vitrolife
59
Media contents G-Series
61
Instruments by Vitrolife
62
Precautions and warnings
64
Correspondence 65
Contents
Introduction
Improving success rates
Vitrolife is dedicated to improve the success rates of Human Assisted Reproduction. Long
term research in reproductive physiology and studies of embryo development has resulted
in the most advanced IVF media products available. This manual describes the use of
Vitrolifes G-Series by Dr. David K. Gardner.
We are well aware that there are many ways of practicing assisted reproductive technology.
The methods we describe below have resulted in high success rates with our products.
The Vitrolife Fertility Products

IVF media
Vitrolife offers IVF media products of the highest efficacy and safety. Every LOT undergoes
rigorous Quality Control testing including mouse embryo assay (MEA) before release to
customers. With every released LOT, a Certificate of Analysis is issued.
Quality Control together with quality assured operations (ISO 13485:2003 and 21 CFR
Part 820:QSR) guarantees LOT-to-LOT consistency. All raw materials used for the manufacturing of Vitrolife Fertility Products are selected as the best available on the world market.
All raw materials are tested and evaluated individually by stringent quality control and MEA
procedures before use in the manufacturing of our media.
Swemed IVF instruments by Vitrolife

Vitrolife also provides a number of instruments for the IVF treatment, such as aspiration
needles, denudation pipettes, pipettes for ICSI and embryo biopsy, and transfer catheters.
Like the media, the instruments undergo a strict quality control and are manufactured in
accordance with the same Quality Standards as the media.
We are certain you will agree that the creation of new human life deserves the best possible environment. We are committed to bringing you the highest quality products.
Introduction
General
Opening your Vitrolife products
When you receive your delivery of Vitrolife products you may notice that they are packaged
in a special way. There are specific reasons for this:
All Vitrolife products are tamper-evident sealed. The packaging ensures that it is
impossible to enter the bottle without visible evidence.
All materials used in the packaging are non-toxic so that nothing may interfere with the
final product. PETG bottles, screw caps, specially designed labels and pharmaceutical
sealing are all part of this tamper evident protocol.
G-Series PLUS media, except G-IVF PLUS, are supplemented with 5 mg HSA/mL.
G-IVF PLUS, is supplemented with 10 mg HSA/mL. All other G5 media are protein-free
(unless otherwise labeled) and need to be supplemented with either G-MM (recombinant
human albumin) or HSA-solution (human serum albumin) according to bottle labels. See
Table on page 7.
Essentials
Wash and disinfect your hands before handling any product.
Take necessary precautions when handling any biological fluid such as blood, follicular
fluid and semen.
Hands on
1 Open all products in a clean laminar air flow (LAF) cabinet.
2 Before bringing bottles into the LAF cabinet, assure the bottles are clean on the
outside. It is recommended that the bottles are wiped with a lint free cloth and ethanol.
3 Identify the product and check the expiry date. Break the tamper-evident seal and
discard.
General
4 Remove the cap and place the cap face down in a sterile petri dish.
5 Remove the desired volume with a sterile non-toxic pipette. Replace bottle cap and
screw on firmly. Do not touch the inner sides of the cap.
6 Keep the media cold as much as possible. Prepare dishes immediately after the bottles
have been removed from the refrigerator. Media bottles should not be left at ambient
temperature for a longer period of time than it takes to prepare dishes or tubes.
Aseptic working techniques means to work in the absence of microorganisms

capable of causing infection or contamination.
CAUTION
Never pour the contents out of the bottle, as the lip of the bottle may not be
sterile once opened.
CO2 equilibration
Optimal pH for culture of human embryos is 7.2- 7.4.
To obtain correct pH, all G-Series media (with the exception of G-MOPS/G-MOPS
PLUS, G-PGD, G-Freezekit Blast and G-Thawkit Blast), should be equilibrated at 6%
CO2. This is a general recommendation that is valid for laboratories located at or near sea
level. For laboratories located at higher altitudes, the CO2 percentage should be increased
by approximately 0.6% CO2 per 1000 meters.
Correct pH must always be verified with pH measurements. See page 10.
Supplementing media with G-MM

or HSA-solution
In this manual the word supplemented is frequently used. Supplemented
means the addition of G-MM or HSA-solution as described in this section, or
G-Series PLUS products.
G-Series PLUS media, G-RINSE, G-GAMETE and EmbryoGlue do not
require albumin supplementation.
General
All other G-Series culture media, not designated PLUS, come protein-free and
need to be supplemented either with G-MM or HSA-solution at the appropriate
concentration as listed in the table below.
When selecting protein supplementation it should be noted that Human Serum
Albumin (HSA) is a blood derived substance and may contain human pathogenic
agents including those not yet known or identified. Thus the risk of transmission
of such infectious agents cannot be completely eliminated when using HSA.
G-MM containing recombinant human albumin in place of HSA may contain
an extremely small amount of yeast antigens (less than 0.15ppm) from the
manufacturing process. Since no animal or human derived raw materials are used
in the manufacturing process of recombinant human albumin, all human or animal
derived infectious agents are proscribed by virtue of the biosynthetic character of
the product.
G-Series media with the exception of G-IVF shall have 5% of either G-MM or HSAsolution added (0.5 mL added to 9.5 mL). The final concentration of G-MM will be 2.5
mg/mL and the final concentration of HSA will be 5mg/mL.
G-IVF shall have 10 % of either G-MM or HSA-solution added (1.0 mL added to 9.0
mL). The final concentration of G-MM will be 5 mg/mL and the final concentration of HSA
will be 10 mg/mL.
Media to be supplemented should be aliquoted into sterile non-toxic tissue culture grade
tubes or flasks. All containers should be pre-rinsed using G-RINSE in order to ensure
there is no particulate matter or toxic substances residing.
Supplementation of G-MOPS, G-1, G-2, G-PGD.

Medium [mL]
G-MM or HSA-solution [mL]
Final volume [mL]
9.5
0.5
10.0
19.0
1.0
20.0
28.5
1.5
30.0
38.0
2.0
40.0
47.5
2.5
50.0
57.0
3.0
60.0
66.5
3.5
70.0

76.0
4.0
80.0
85.5
4.5
90.0
95.0
5.0
100.0
General
Supplementation of G-IVF
Medium [mL]
G-MM or HSA-solution [mL]
Final volume [mL]
9.0
1.0
10.0
18.0
2.0
20.0
27.0
3.0
30.0
36.0
4.0
40.0
45.0
5.0
50.0
54.0
6.0
60.0
63.0
7.5
70.0

72.0
8.0
80.0
81.0
9.0
90.0
90.0
10.0
100.0
G-FreezeKit Blast and G-ThawKit Blast require the following

supplementation (please read pages 50 and 52):
9.0 mL Freeze/Thaw solution + 1.0 mL G-MM or 1.0 mL HSA-solution
The cryopreservation solutions can be supplemented directly into the dishes. Simply place
900 L of each cryopreservation solution into separate wells + 100 L of G-MM or 100L
HSA-solution.
It is recommended only to supplement the volume of medium expected to be
used in one day.
Recommended use of G-MOPS /G-MOPS PLUS

G-MOPS/G-MOPS PLUS are intended for handling of gametes and embryos outside of
the CO2 incubator.
Essentials
In order not to subject oocytes and embryos to unnecessary stress when working outside of
the CO2 incubator, it is very important to work as quickly as possible. As the lid of the dishes
must be removed when moving oocytes and embryos between dishes and when performing
ICSI, pH as well as temperature and osmolality may change after some time. This is valid for
all media including G-MOPS/G-MOPS PLUS.
General
Importance of pH
G-MOPS/G-MOPS PLUS are MOPS buffered media and must be used at +37C in
an air atmosphere. Do not put this medium in a CO2 environment as the pH will go down
below the specification range. Furthermore, do not use paraffin oil equilibrated in a CO2
environment when covering G-MOPS/G-MOPS PLUS.
Importance of Temperature
Oocytes and embryos must be kept at +37C at all times.
To ensure the temperature of G-MOPS/G-MOPS PLUS inside of a tube/dish is +37C,
it is recommended to validate the procedure using a certified thermometer that is placed
inside of a tube/dish containing a sample amount of G-MOPS/G-MOPS PLUS, in the
warming block (tube) or on a heated stage (dish). The goal is to ensure the G-MOPS/GMOPS PLUS is +37C before use. Individual pieces of warming equipment should be
adjusted to attain this goal.
Importance of Osmolality
To avoid changes in osmolality, tubes must be filled as much as possible and tightly capped.
Dishes should be covered with oil or kept with the lid on when not in use.
G-MOPS for oocyte aspiration

G-MOPS for oocyte aspiration should not to be supplemented. In order to ensure the
G-MOPS is appropriately warmed to +37C before use, pre-warm the medium by placing
a tightly sealed bottle of G-MOPS into a warming incubator without CO2. The bottle must
be tightly capped to avoid changes in osmolality. It can take up to 4 hours for a bottle of
G-MOPS to reach +37C. Each time the bottle is removed from the warming incubator
and used, ensure the cap is tightly replaced. Discard any G-MOPS that is warmed and
not used within the recommended time frame, i.e. the same day if the medium is stored in a
tightly capped tube until use or after 60 min if the medium volume is minimum 1 mL in a dish
and not covered with oil. A dish covered with oil can be used for up to 2hours provided that
the appropriate temperature of the heating stage is maintained.
G-MOPS for gamete and embryo handling

G-MOPS for gamete and embryo handling must to be supplemented. In a laminar flow
hood pipette G-MOPS/G-MOPS PLUS into non toxic sterile test tubes. If G-MOPS is
used supplement with the appropriate amount of G-MM or HSA-solution. The tube must
be filled and tightly capped to avoid changes in osmolality. Place the tube in a warming
incubator without CO2 or in an adequately calibrated warming block. Pipette pre-warmed
G-MOPS/G-MOPS PLUS into a pre-warmed culture dish. To avoid changes in osmolality
use the medium as soon as possible after preparation, i.e. 60 minutes if the dish is not
covered with oil (minimum 1 mL of medium) or 2 hours if the dish is covered with oil.
General
Pipette pre-warmed G-MOPS/G-MOPS PLUS into a culture dish placed in a

warming incubator without CO2 or on a heated stage, with the lid on. To avoid
changes in osmolality, cover with oil or use the dish within 60 minutes after
preparation. This statement is valid for a volume of 1 mL or higher. To ensure
that the temperature inside of the culture dish is +37C it is recommended
to validate the procedure using a certified thermometer that is placed inside
of a culture dish with G-MOPS/G-MOPS PLUS on a heated stage. The
temperature setting of the heated stage needs to be adjusted to achieve the
correct +37C of the medium in the culture dish.
IMPORTANT
Ensure that no G-MOPS/G-MOPS PLUS is transferred to the culture dishes
by introducing washing steps between the G-MOPS dish and the culture
dish. The washing procedure should include at least two steps of 1 mL culture
medium per step. The wash dishes should be changed after 5 oocytes or
embryos.
How to measure pH
Introduction
Measurement of pH with a glass membrane electrode and a modern instrument is a
straightforward process. There are however several pitfalls to be avoided when the highest
possible accuracy needs to be achieved. This is especially the case if pH must be measured
in solutions with dissolved gas or with high protein content.
Essentials
Use a semi-micro or a micro electrode with a built in reference electrode and
temperature measurement.
Set the instrument for automatic temperature compensation.
Use fresh buffers with at least one having a pH close to that of the tested solution.
Change the buffer between every test.
Confirm the proper function of the electrode daily by testing slope and offset.
Change the electrode as soon as it starts to deteriorate. A pH-electrode rarely lasts for
longer than 12 months and may often have to be replaced every second or third month.
Store the electrode in a neutral buffer or in a special storage solution.
Calibrate the instrument before each sequence of testing.
10
General
Hands on suggested procedure of pH measurement

(The exact procedure may vary depending on instrumentation and the nature of the sample)
1 Take the electrode from the storage solution, rinse it and place it in pH 7.00 buffer.
Remove the plug or tube that blocks the air inlet to the electrode.
2 Let the electrode stand in the pH 7.00 buffer for between one and two hours. This time
may be reduced if the electrode is stored in pH 7.0 buffer over night.
3 Calibrate the electrode using two fresh buffers, one close to pH 7.00 and one at e.g. pH
10.0.
4 Verify that the slope and the offset of the electrode are within the specified limits of
the electrode manufacturer. We recommend that the slope should be between 95 and
102% of the theoretical value, and that the offset at pH 7.00 should not differ more than
4 mV from the value acquired during the previous calibration.
5 Verify that the calibration was performed properly by testing a buffer with a pH of
approximately 8.00. Compare the measured and the specified pH at the actual test
temperature. The measured pH may not differ from the specified pH by more than
0.05 pH units.
6 Place the electrode in distilled water at approximately the same temperature that the
sample will have when it is tested.
7 Equilibrate the sample at the temperature and gas composition that is required.
Equilibration with CO2 may take up to 16 hours!
8 Take the electrode from the distilled water; wipe the tip of the electrode briefly with
a soft clean tissue to remove the water. Place the electrode immediately in the
equilibrated media. The glass bulb and the contact point of the reference electrode
PH
measure
must be covered with the equilibrated media, see a) and b) below.
a)
a) Sintered disk window
b)
b) Sleeve joint
SWIMUP C
a)
b)
General
11
9 Take a reading from the pH instrument as soon as the pH is stable. If the test media
has been equilibrated with CO2 the entire test procedure must be completed within 30
seconds from the removal of the media from the incubator. If the test takes longer time
than this, CO2 will dissipate from the sample into the atmosphere and pH in the medium
will rise.
10 As an extra precaution a control medium with known pH at the conditions at hand may
be tested in parallel with the unknown sample. This enables a confirmation to be made
that the entire test procedure was performed correctly.
11 Register all data from the test, including the calibration data, in appropriate records or
log-books.
IVF and Culture system

We are aware that there are many options to choose from when considering the culture
system for IVF. This manual provides a suggested methodology that has been proven successful for our products.
Essentials
Choose your culture systems for their efficacy, simplicity and reproducibility.
Organise yourself and prepare in advance.
Use only non-toxic sterile disposables that are subject to quality control for embryo
culture and keep a log of all items and procedures performed.
Adhere to your protocols to ensure consistency.
Ensure that all of your equipment is correctly maintained, and regularly checked and
calibrated.
All procedures should be performed in a clean, dedicated work environment
(LAF cabinet).
Use aseptic working techniques.
Qualify all operators for handling media and keep training protocols consistent.
Always wear non-toxic gloves when handling biological fluids.
Check the identity of the patient and label all materials before proceeding.
12
General
Sperm
Preparation
Prior to being used for IUI, IVF and ICSI, motile sperm cells are separated from the seminal
plasma, dead sperm cells and other cells. This can be done by different procedures.
The decision of which semen preparation method to use is based on the patient history, previous
semen analyses, as well as an examination of the present sample. Another consideration is whether
fertilisation will be achieved by IVF or ICSI. In IVF you will need more sperm for insemination. The
preparation methods select sperm based on their motility, ideally selecting only live sperm, or on
their density, ideally selecting only mature sperm. If the sperm count and motility are adequate,
migration (swim-up) is suitable. If semen quality is poor and includes large numbers of other cells,
density gradient centrifugation is preferred. Recovery of sperm is more effective using the gradient
centrifugation method rather than using the swim-up procedure, with respect to total yield. However,
in some instances percent motility can be higher in sperm prepared by swim-up.
Essentials
The sperm preparation should be performed in a clean aseptic work area. Non-toxic,
non-powdered gloves and protective eye glasses should be worn while handling semen
samples.
All samples should be collected in appropriate sterile, non-toxic vessels. It is
recommended that the semen sample be collected not more than one hour before
preparation. The semen sample should be protected from cold and heat.
All laboratory procedures, including a thorough identification protocol of the patient
should be followed.
Sterile, non-toxic tubes, needles and pipettes should be washed with G-RINSE before
use for the preparation of sperm.
Sperm Preparation
13
Swim-up method (migration)

Hands on
This method is to be used for the semen samples with a good sperm count and motility.
1 Allow approximately 20 minutes for liquefaction of semen. If the sample does not liquefy,
you may need to pass it through a 23 gauge needle or a non-toxic sterile narrow Pasteur
pipette.
Liquify for 20 minutes
Liquify for 20 minutes
2 Make a microscopic assessment of the sperm sample to confirm the optimal method for
processing the sperm.
PH measure
a)
b)
3 Mark a test tube with patient ID. More than one tube can be set up if there are concerns
about semen quality.
4 Pipette 1.0 mL of semen into a rinsed tube. Make up 2-4 tubes depending on semen
volume. Carefully overlay 2.0 mL of pre-equilibrated supplemented G-IVF/G-IVF PLUS.
Place the swim-up tube in an angled position in the incubator at +37C and 6 % CO2 for
SWIMUP C
3060 minutes.
a)
a)
b)
b)
Incubate in 37C, 6 % CO2 for 3060 minutes.

a) G-IVF / G-IVF PLUS b) Semen
SWIMUP D/E
14
Sperm Preparation
b)
5 Aspirate the top medium without touching the underlaying semen and transfer to a clean
SWIMUP
D/E
tube.
Add 5.0
mL of equilibrated supplemented G-IVF/G-IVF PLUS, mix and centrifuge
for 10 minutes at 300600 g.
Remove top medium. Dilute with G-IVF / G-IVF PLUS. Centrifuge.
6 Discard the supernatant and re-suspend the pellet in 5.0 mL of equilibrated

supplemented G-IVF/G-IVF PLUS and repeat the centrifugation at 300600 g for 10
DENS A GIII
min.
A
Remove
supernatant.
Repeat
wash.
a)
b)
c)
Discard supernatant. Repeat wash.
7 Discard the supernatants and combine all pellets. Re-suspend the pellets in 0.51.0 mL
DENS
B,C,Dsupplemented
GIII
of
equilibrated
G-IVF/G-IVF PLUS, depending on sample quality.
B
Resuspend in
small volume
G-FERT.
Resuspend
a) in small volume G-IVF / G-IVF PLUS.
8 Determine motility and concentration of spermatozoa in the washed sample.

9 Dilute the washed sample with equilibrated supplemented G-IVF/G-IVFPLUS to a
PREPCULT
A,Bof 75 000200 000 motile sperm/mL.
final
concentration
10 Prepare rinsed insemination
dishes with 0.5-1.0 mL ofa)sperm suspension and
a)
A
equilibrate at +37C and 6 % CO2 for at least 2 hours.
Alternatively: Add equilibrated sperm suspension to equilibrated dishes with the

oocytes already present.
If oil overlay is used, droplets of at least 100 L volume are recommended.
b)
b)
MICRO A,B
A
a)
f)
Sperm Preparation
b)
a)
b)
15
PH measure
Density
gradient centrifugation method
This method can be used to wash all samples of sperm regardless of quality.
SpermGrad is a solution of silane coated, colloidal silica particles in an isotonic balanced salt
solution. By usinga)
different dilutions of SpermGrad, solutions
b) of different densities are obtained.
Layering these solutions of different densities carefully in a centrifuge tube creates a density gradient. Cells and other particles with different buoyant densities will sediment until they reach a solution
with higher density. Centrifugation accelerates this sedimentation. Commonly, a two-step gradient
of 90 % and 45 % SpermGrad is used. Since mature sperm with tightly packed DNA have a higher
density than 90 % SpermGrad, they sediment through this layer and are found at the bottom of the
tube, whereas other cells, including immature and dead sperm, stop sedimenting at the 90 % or 45
%SWIMUP
interface. YouCcan use either SpermGrad RTU solutions already diluted to 90% (Upper Layer)
and 45% (Lower Layer) or SpermGrad 100% stock solution, should you prefer to make your own
dilutions. The stock solution must be diluted in G-IVF/G-IVF PLUS into appropriate concentrations. G-IVF should be supplemented with either G-MM or HSA-solution. G-IVF/G-IVF
PLUS must be equilibrated at +37 C and 6% CO2 before use.
Hands on a)
b)
1 If you use SpermGrad RTU, continue to paragraph 2. If you use SpermGrad stock
solution:
Mix SpermGrad with supplemented G-IVF/G-IVF PLUS in separate tubes to obtain
90SWIMUP
% and 45 D/E
% stock solutions. For 90 % stock solution, mix 9.0 mL SpermGrad with
1.0 mL supplemented G-IVF. For 45 % stock solution, mix 4.5 mL SpermGrad with
5.5 mL supplemented G-IVF. Mix the solutions thoroughly and store in sterile non-toxic
tubes or sterile tissue culture flasks. Label and refrigerate until use. Always use a sterile
non-toxic pipette to aliquot amounts needed for individual sperm preparations. Stock
solutions should be labeled with the date and kept for recommended time frames (see
expiry on bottles). Before use, allow the solutions to reach ambient temperature.
2 The density gradient should be layered in 2 to 4 sterile and rinsed conical non-toxic
centrifuge tubes (depending on the volume of the semen sample) marked with patient
ID. Pipette 1.5 mL of 90 % solution into the tube first and then slowly pipette 1.5 mL of
45 % solution on top of it. Finally, 1.0 mL of the semen is gently layered on the top (A).
Make up 2-4 gradient tubes. Up to 2 mL of semen can be layered on top of the gradient.
If the
semen
sample is of normal quality reduce the semen volume. Adding too much
DENS
A GIII
semen will result in poor separation.
A
a)
b)
c)
A. Layer gradient solutions and semen in conical centrifuge tube. Spin 300600g / 20 minutes.
a) Semen b) 45 % c) 90 %
DENS B,C,D GIII
16
Sperm Preparation
B
Vitrolife
G-Series Manual 6.0
D
DENS A GIII
3 The tubes are then centrifuged for 1020 minutes at 300600g.
A
4 Remove the two top layers and take care not to leave any residues on the tube wall (B).
Transfer the sperm pellets with as little of the 90 % solution as possible to a sterile
a)
conical rinsed tube with 5 mL of equilibrated supplemented G-IVF/G-IVF PLUS.
b)
c) for 10 minutes at 300600g.

5 Centrifuge
6 Aspirate and discard the supernatants and repeat the wash (C). After the second wash,
combine pellets and re-suspended in 1 mL of equilibrated supplemented
B,C,D
DENS
GIII (D). The washed sample is then assessed for motility and
G-IVF
/G-IVF PLUS
concentration.
B
a)
B. Remove top layers
and transfer pellet
to clean tube.
a)
Pellet
PREPCULT
A,B
C. Wash pellet with

G-IVF/G-IVF PLUS.
REPEAT.
D. Resuspend pellet
in small volume
of G-IVF/G-IVF PLUS.
7 Dilute the washed sample with equilibrated supplemented G-IVF/

a)
a)
A
B
G-IVF
PLUS to a final concentration of 75
000200 000 motile sperm/mL.
8 Prepare rinsed insemination dishes with 0.51.0 mL of sperm suspension and
equilibrate at +37 C and 6 % CO2 for at least 2 hours.
b)
b)

ItMICRO
is recommended
to inseminate in a volume of 0.5-1.0 mL without oil overlay. If oil
A,B
overlay is used, droplets of at least 100 L volume are recommended.
A
a)
f)
b)
a)
c)
b)
d)
c)
e)
d)
B
b)
a)
d)
c)
MICRO SETUP A,B
Sperm Preparation
17
18
Sperm Preparation
IVF-ET
Vitrolife has developed culture media for the individual stages of embryo development.
G-IVF/G-IVF PLUS should be used for in vitro fertilisation. For cleavage and culture of early embryos G-1/G-1 PLUS should be used. For culture from day 3 up to the blastocyst stage, G-2/G-2
PLUS should be used. After ICSI, embryos should be placed directly into G-1/G-1PLUS.
Irrespective of stage, embryos should be transferred to the uterus using EmbryoGlue or supplemented G2 or G-2 PLUS.
Follicle aspiration
The purpose of the oocyte collection procedure is to collect as many oocytes as quickly as
possible and to minimise the exposure of those oocytes to non-physiological conditions.
Important parameters are temperature, osmolality and pH. Any deviations from physiologic
conditions may have deleterious effects on the ability of the oocyte to fertilise normally and
achieve normal preimplantation development.
Essentials
Day 0 is defined as the day of oocyte collection.
Identify the patient and check ID label on culture dishes and pipettes before proceeding.
Ensure that all surfaces are warmed and that all materials that may come in contact with
the oocytes are sterile, non-toxic and of tissue culture quality, preferably mouse embryo
tested.
Oocyte aspiration is performed with the use of an ultrasonically guided needle with a
double or single lumen. For recommended aspiration needles, see Swemed Instruments
by Vitrolife page 62, Follicle Aspiration needles, V-Tip. Negative pressure is typically
applied to the needle lumen by a regulated aspiration pump.
High or uncontrolled negative pressures may damage oocytes.
IVF-ET
19
Pre-warmed G-MOPS is used as the rinsing fluid for the procedure.

G-MOPS is a modified G-1 medium, buffered with MOPS and sodium bicarbonate to
maintain pH at room atmosphere.
For collecting aspirates and follicle flushing pre-warmed G-MOPS can be used
protein-free due to the high protein content of follicular fluid.
For washing of oocytes prior to incubation in supplemented G-IVF/G-IVFPLUS, prewarmed supplemented G-MOPS/G-MOPS PLUS should be used.
G-GAMETE equilibrated at +37C and 6% CO2 can be used as an alternative to
supplemented G-MOPS/G-MOPS PLUS.
It is advised to prepare one wash dish for a maximum of five follicles.
Hands on
1 Pre-equilibrate G-RINSE at +37C in an atmosphere of 6 % CO2.
2 Warming of G-MOPS PLUS for oocyte wash: pipette the medium in rinsed tubes.
Tightly cap the tubes and place them in a warming incubator without CO2 at +37C.
3 Warming of un-supplemented G-MOPS for follicle flushing: pipette the medium in
rinsed tubes. Tightly cap the tubes and place them in a warming incubator without CO2
at +37C.

Before use, ensure that the temperature of the media is +37C.
4 Rinse the needle lumen and tubing using G-RINSE and discard the rinsed fluid.
5 The follicles may be aspirated individually or collectively. The aspirated follicular fluid
must be collected in a rinsed sterile non-toxic tissue culture tube. G-MOPS can be
supplemented with quality tested, pharmaceutical grade Heparin (2.510.0 units/mL)
to reduce clotting of the follicular aspirates containing blood.
6 The follicle aspirates should be collected in sterile non-toxic tissue culture disposable
tubes and should be examined by the laboratory immediately. If they cannot be
examined directly, the tubes should be tightly sealed and kept at +37C.
Oocyte identification
Essentials
wear non-toxic gloves
identify the patient before proceeding
work rapidly to avoid cooling
20
IVF-ET
Hands on
1 Follicle aspirates should be kept at +37C. The use of a test tube heating block next
to the microscope can facilitate this. Follicle aspirates are microscopically examined in
sterile tissue culture Petri dishes.
2 Should it be necessary to flush a follicle, G-MOPS pre-warmed to +37C should be
used.
3 Transfer the follicular aspirates to an empty dish. Identify the oocytes, pre-rinse a sterile
pipette with supplemented G-MOPS/G-MOPS PLUS, and immediately remove the
oocytes from the follicular fluid and possible blood contamination.
4 Rinse the oocytes first in pre-warmed supplemented G-MOPS/G-MOPS PLUS,
and then in equilibrated supplemented G-IVF/G-IVF PLUS. The washing procedure
should include at least two steps with 1.0 mL of G-IVF/G-IVF PLUS in each step.
Transfer the oocytes to the dishes with equilibrated supplemented G-IVF/G-IVF PLUS
and return the dishes to the incubator immediately.
Oocyte culture and fertilisation

Essentials
Crucial considerations for gamete handling and fertilisation are temperature, pH and
osmolality.
Temperature loss occurs rapidly and is related to the amount of time a culture vessel
stays out of the incubator and whether the surface is heated, e.g. a heated microscope
stage.
Changes in osmolality depend on volume, temperature and presence or absence of oil
overlay. Changes in osmolality will occur slowly and is often an undetected parameter.
The culture system should be correctly humidified and preferably under oil.
Changes in pH occur rapidly and, like temperature, is related to the time the culture
vessel stays out of the incubator and the time of exposure to air.
Ensure that all surfaces are warmed and that all materials that may come in contact with
the oocytes are sterile, non-toxic and of tissue culture quality, preferably mouse embryo
tested.
IVF-ET
21
Insemination
Hands on
1 Transfer the oocytes to insemination dishes containing 75 000200 000 sperm/mL and
leave at +37C and 6 % CO2 over night.


Several oocytes may be inseminated in the same vessel. If oil overlay is used, droplets of
at least 100 L volume are recommended.
Fertilisation assessment
Essentials
IVF inseminated oocytes are checked for fertilisation approximately 1520 hours after
addition of sperm (day 1). ICSI oocytes may be observed 1218 hours after injection,
as pronuclei (PN) may appear earlier.
Fertilised oocytes are sensitive to temperature and pH variations and it is important to
minimise these changes by using pre-warmed supplemented G-MOPS/G-MOPS
PLUS as a handling medium outside of the incubator.
G-MOPS/G-MOPS PLUS contains the buffer MOPS to stabilise pH during handling
in room atmosphere and does not require equilibration in a CO2 atmosphere prior to
use. Rather, this medium is designed to maintain a pH of 7.2 7.4 outside of a CO2
atmosphere.
G-GAMETE can be used as an alternative to supplemented G-MOPS/G-MOPS
PLUS. G-GAMETE should be equilibrated at +37C and 6 % CO2 before use.
It is recommended to heat all microscope stages and to work rapidly. To ensure that the
temperature inside of the dish is correct, please verify by using a certified thermometer
or a calibrated digital thermometer with a thermocouple that can be secured to the
stage and to the inside of the dish.
Hands on fertilisation assessment

1 Transfer the oocytes to a dish with pre-warmed supplemented G-MOPS/
G-MOPS PLUS.
2 Remove cumulus and corona cells from oocytes at +37C using a denudation pipette,
see Swemed Instruments by Vitrolife page 62. The cumulus and corona cells are often
well dispersed by the hyaluronidase from the sperm. If the cumulus cells are not well
22
IVF-ET
dispersed, needles may be used to attentively tease the oocyte out of the corona cells.
Cells need only be removed to the extent that polar bodies and pronuclei can be clearly
observed.
3 Observe microscopically and record the number of pronuclei, polar bodies and the
possible presence of a germinal vesicle. It is recommended to make these observations at high magnification (minimum 200X) on an inverted microscope with Nomarski
or Hoffman optics. It is difficult to be accurate in the assessment of fertilisation if lower
magnifications are used. Only embryos derived from normally fertilised oocytes (2PN)
should be considered for embryo culture and transfer. Non-fertilised or degenerated
oocytes, oocytes with only 1 PN, and oocytes with more than 2 PN should be removed
from culture. Pro-nuclear scoring, see the following page.
4 After assessment, the fertilised oocytes should be rinsed thoroughly in several droplets
of equilibrated supplemented G-1/G-1 PLUS and then cultured in equilibrated supplemented G-1/G-1 PLUS, preferably under oil.
Scoring of human pronuclear embryos

From: Scott L, Alvero R, Leondires M and Miller B. 2000. The morphology of human pronuclear embryos is
positively related to blastocyst development and implantation. Hum Repr 15, No 11, pp 2394-2403
Essentials
Zygote morphology has been related to blastocyst development, implantation and
pregnancy
The scoring system is used to assess the developmental capacity of zygotes and is
based on the morphology of the two pronuclei (2PN)
Pronuclear scoring can be combined with scoring on Day 3 and/or blastocyst scoring
It is recommended to transfer not more than two embryos in order to avoid the
complications of multiple pregnancies.
Hands on
Scoring of zygotes should be performed on an inverted microscope while maintaining
physiological pH and temperature.
Scoring should be performed 15-20 hours after insemination or 12-18 hours after
ICSI, since the pronuclei of injected oocytes may appear as well as disappear earlier
than for inseminated oocytes.
It is recommended to use supplemented G-MOPS /G-MOPS PLUS or
G-GAMETE for this procedure.
IVF-ET
23
SWIMUP D/E
Scoring System
The nuclear size should be equal
The nucleoli need to be aligned at the pronuclear junction
The number of nucleoli should be between three and seven per nucleus with no more
than one nucleolus difference between the nuclei
The nucleoli should be equal in size
To obtain optimal pregnancy and implantation rates, embryos of good morphological
quality that showed pattern Z1 at the PN stage should preferably be chosen for transfer.
Pattern
DENS A GIII
Z1
Z2
Z3
Z4
a)
b)
c)
Z1
Z2
Z3
Choosing
the
culture
system
DENS B,C,D GIII
Z4
There are essentially two culture systems to choose from for culture of gametes and
B
D
C
embryos:
A
Large volumes (0.5 mL 1.0 mL) in tubes or wells, with or without oil
Small volumes (droplets 0.05 mL 0.1 mL) under oil

a)
The surface areas of systems without oil are large enough for the osmolality to change over
several days. We therefore recommend the use of oil as a protection against changes in
temperature and osmolality and as a barrier to dust particles and any microorganisms from
the atmosphere. Paraffin oil is chosen for its viscosity and its high purity. OVOIL is pharmaPREPCULT A,B
ceutical grade light paraffin oil, sterilised by filtration using a 0.22 m pore size sterile filter.
a)
a)
b)
b)
A. Multi-well with OVOIL

a) OVOIL
b) Medium
B. Droplets under oil

a) OVOIL
b) Medium droplets 10100 L
MICRO A,B
A
a)
f)
24
IVF-ET
b)
a)
c)
b)
d)
Preparation of culture system

Embryo cultures should ideally be performed in 6 % CO2 and 5 % O2 for all media. This
gas environment can be created using either a tri-gas incubator or a modular incubator
chamber/isolette and fed with a gas mix (6 % CO2, 5 % O2 and 89 % N2). There are
significant benefits to using a low oxygen environment. Should it not be possible to use a
reduced oxygen environment, use 6 % CO2 in air atmosphere.
Hands on
1 All culture dishes (wells or tubes) should be rinsed with G-RINSE and prepared in
advance of the procedure.
2 In the afternoon of the day of oocyte retrieval, label a micro-droplet dish with ID of the
patient. Using a pre-rinsed sterile tip, fill an appropriate number of micro-wells up to
25L of supplemented G-1/G-1 PLUS. Immediately cover the drops with OVOIL to
avoid evaporation. Do not prepare more than 1 dish at the time.

Alternatively, label a 40 mm culture dish with ID of the patient. Using a pre-rinsed sterile
tip, place droplets of up to 50L of supplemented G-1/G-1 PLUS at the bottom of
the dish. Immediately cover the drops with OVOIL to avoid evaporation. Do not prepare
more than 1 dish at the time.
To achieve round standing droplets, you can do like this: place 6x25 L droplets of
supplemented G-1/G-1 PLUS at the bottom of a 40 mm culture dish. Immediately
cover the drops with OVOIL to avoid evaporation. Using a new tip for each drop, first
rinse the tip and then take a further 25 L of medium to each original drop. Do not prepare more than 1 dish at the time. This technique allows the droplets to stand up rather
than flatten out.
3 Immediately place the dish in the incubator at 6 % CO2 and +37C. Gently remove the
lid of the dish and set at an angle on the side of the plate. This will ensure proper equilibration of the dish.
Dishes must equilibrate in the incubator with a semi-opened lid for a minimum
of 6 hrs (this is the minimal measured time for the media to reach correct pH
under oil) and for a maximum of 18 hours.
IVF-ET
25
Embryo culture
Essentials
Crucial considerations for embryo culture are temperature, pH, and osmolality.
Temperature loss occurs rapidly and is related to the amount of time a culture vessel
stays out of the incubator. It is recommended to work on heated surfaces, i.e. the
microscope stage.
Changes in osmolality will occur slowly and is often an undetected parameter. The
culture system should be correctly humidified and preferably under oil.
Changes in pH occur rapidly when medium is exposed to air. Ensure the culture dishes
are out of the incubator for the minimal time period possible.
High implantation and pregnancy rates can be achieved by transfer of blastocysts.
Blastocyst culture and transfer will not rescue a deficient day 2 / day 3 IVF-ET program.
Cultivation of blastocysts for transfer and cryopreservation requires rigorous control of
laboratory conditions.
A sufficient number of incubators is a prerequisite for successful extended culture.
Ideally, two incubators should be used for no more than 5 retrievals per week. One of
the incubators should be used for embryo culture, while the second should be used for
the pre-equilibration of the media dishes. Culture incubators should not be used for preequilibration of media to minimize the number of door openings occurring.
Hands on: Culture of Cleavage Stage Embryos

Day 1 Culture in G-1/G-1 PLUS
1 Once the cumulus is removed, all manipulations should be performed using a pulled
Pasteur pipette, see Swemed Instruments by Vitrolife page 62, Transfer pipette. It is
important to use a pipette containing a tip with a diameter that is slightly larger than that
of the embryo. To avoid damage, it is very important that the tip is not smaller than the
embryo. For example, for embryos on day 1 to 3, a pipette tip of 150 to 200 m would
suffice. Using the appropriate size tip minimises the volumes of culture medium moved
with each embryo, which typically should be less than one microlitre.
Such attention to volume manipulation is a pre-requisite for successful culture.
2 Following removal of the cumulus cells and assessment of fertilisation, pronucleate

embryos are transferred to a centre well dish and washed in pre-warmed supplemented
G-MOPS/G-MOPS PLUS or in equilibrated G-GAMETE. Washing entails picking
up the embryo 23 times in a minimal volume and moving it around within the well.
Embryos should then be washed successively in at least two drops in the culture dish.
26
IVF-ET
More extensive washing will reduce the risk of transferring medium from the old dish to
the fresh one. Residues of MOPS-buffer in the culture droplets may decrease pH below
specification.
3 Place the embryos in the equilibrated supplemented drops of G-1/G-1 PLUS. As

a precautionary measure, prepare two culture dishes if the patient has more than 10
embryos. Return the dish to the incubator immediately. It is advisable to culture embryos
in at least groups of 2. For example, for a patient with 6 embryos it is best to culture in 2
groups of 3 rather than 4 and 2 or 5 and 1.
4 On day 3, embryos can be transferred to the uterus in equilibrated EmbryoGlue or in

equilibrated supplemented G-2/G-2 PLUS. Alternatively, on day 3, embryos can be
transferred to equilibrated supplemented G-2/G-2 PLUS for further culture to the
blastocyst stage.
Hands on: Culture of Blastocyst Stage Embryos

Day 3 Culture in G-2 /G-2 PLUS
1 In the morning of day 3, label a micro-droplet dish with ID of the patient. Using a prerinsed sterile tip, fill an appropriate number of micro-wells up to 25 L of supplemented
G-2/G-2 PLUS. Immediately cover the drops with OVOIL to avoid evaporation. Do
not prepare more than 1 dish at the time. Immediately place the dish in the incubator at
6% CO2 and +37C. The minimum equilibration time is 6 hours before use. If the patient
has more than 10 embryos make up two culture dishes.

Alternatively, label a 40 mm culture dish with ID of the patient. Using a pre-rinsed sterile
tip, place droplets of up to 50L of supplemented G-1/G-1 PLUS at the bottom of
the dish. Immediately cover the drops with OVOIL to avoid evaporation. Do not prepare
more than 1 dish at the time.
To achieve round standing droplets, you can do like this: place 9x25 L droplets of
supplemented G-2/G-2 PLUS at the bottom of a 40 mm culture dish. Immediately
cover the drops with OVOIL to avoid evaporation. Using a new tip for each drop, first
rinse the tip and then take a further 25 L of medium to each original drop. Do not prepare more than 1 dish at the time. This technique allows the droplets to stand up rather
than flatten out. Immediately place the dish in the incubator at 6% CO2 and +37C.
The minimum equilibration time is 6 hours before use. If the patient has more than 10
embryos make up two culture dishes.
Alternatively, prepare the dishes for blastocyst culture in the afternoon of day 2 and
equilibrate at 6 % CO2 and +37C over night.
2 For each patient, set up one wash dish of pre-warmed supplemented G-MOPS/
G-MOPS PLUS or equlibrated G-GAMETE per 10 embryos. Place 1 mL of
G-MOPS/G-MOPS PLUS/G-GAMETE into the well of a centre well dish. Place
2mL of G-MOPS/G-MOPS PLUS/G-GAMETE into the moat of the dish. Place on a
heated stage.
IVF-ET
27
3 For each patient, set up one sorting dish. Place 1 mL of G-MOPS/G-MOPS PLUS/
G-GAMETE into the well of a centre well dish. Place 2mL of G-MOPS/G-MOPS
PLUS/G-GAMETE into the moat. Place on a heated stage.
G-MOPS should not be placed in a CO2 incubator, but rather pre-warmed in a
sealed container using an incubator without CO2 or a tube warming block.
4 In the afternoon of day 3, the embryos that were assessed for cleavage in the morning are transferred to equilibrated supplemented G-2/G-2 PLUS. The embryos will
remain in the supplemented G-2/G-2 PLUS in 6 % CO2 and +37C until the assessment for transfer on the morning of day 5. If there is concern about the embryo cleavage
rate, nursing and medical staff should be notified.
5 Wash embryos in the wash dish (this step is crucial to remove the EDTA in G-1/
G-1 PLUS). Washing entails picking up the embryo in a minimal volume of media 23
times and moving it around within the well. After washing, transfer embryos to the sorting dish and group like embryos together. Rinse through the wash drops of the equilibrated supplemented G-2/G-2 PLUS in the culture dish and again place up to five
embryos in each of the culture drops. Return the dish to the CO2 incubator immediately.
6 On day 4, prepare three dishes of supplemented G-2/G-2 PLUS. Label the dishes
Transfer, Freeze and Hold (for culture to day 6). The dishes should be equilibrated
overnight at +37C and 6 % CO2. Dishes should be pre-equilibrated for no less than 6
hours prior to use.
7 Transfers should be performed either in equilibrated EmbryoGlue or in equilibrated

supplemented G-2/G-2 PLUS. Equilibrate transfer dishes at +37C and 6 % CO2 at
least 6 hours prior to use.
8 On day 5, embryos should be evaluated and one or two top scoring blastocysts selected for transfer. Good quality blastocysts not transferred can be cryopreserved. Should
an embryo not have formed a blastocyst by day 5, it should be cultured in a fresh drop of
equilibrated supplemented G-2/G-2 PLUS for 24 hours and assessed on day 6.
9 Move the selected blastocyst(s) to supplemented G-2/G-2 PLUS and leave at +37C
and 6 % CO2 until 1030 minutes before transfer.
Embryo assessment
Essentials
Embryos may be transferred to the uterus on day 2 (4048 hours post insemination)
or day 3 (6674 hours post insemination), or at the blastocyst stage on day 56
(120144 hours post insemination).
28
IVF-ET
The embryo morphology is assessed as close to the time of transfer as possible.

The early embryos are assessed for total number of blastomeres, regularity of the
blastomeres, presence and percentage of fragmentation, granularity of blastomeres,
and cleavage rate. For assessment of blastocysts, see Scoring criteria for human
blastocysts on the following pages.
The embryo(s) chosen for transfer are placed into a fresh dish of equilibrated
EmbryoGlue or equilibrated supplemented G-2/G-2 PLUS, irrespective of stage.
Scoring criteria for human blastocysts

From: Gardner DK and Schoolcraft WB (1999) In-vitro culture of human blastocysts. in Towards
Reproductive Certainty: Infertility and Genetics Beyond 1999. eds. Jansen, R. and Mortimer, D.
Parthenon Press, Carnforth, pp 378-388.
Essentials
The scoring system is used to assess the developmental capacity of blastocysts
based on their morphological appearance, and thereby enable selection for transfer or
cryopreservation.
Scoring of blastocysts should be performed on an inverted microscope while
maintaining physiological pH and temperature.
The majority of the blastocysts should develop by day 5 (see below).
The blastocysts selected for transfer (maximum two) should be placed in the dish
labeled Transfer, and the blastocysts selected for cryopreservation should be placed
in the dish labeled Freeze.
It is recommended to transfer not more than one blastocyst in order to avoid the
complications of multiple pregnancies. Preference should be given to blastocysts
graded as 3 and higher. Select the best scoring blastocysts preferentially, i.e. AA.
Hands on
1 Blastocysts are given an alphanumeric score from 1 to 6, based on their degree of
expansion and hatching status and two letter scores for inner cell mass (ICM) and trophectoderm.
2 The initial phase of the assessment can be performed on a dissection microscope.

3 The second step in scoring the blastocysts should be performed on an inverted microscope. For blastocysts graded as 3 to 6 (i.e. full blastocysts onwards) the development
of the inner cell mass (ICM) and trophectoderm can then be assessed.
IVF-ET
29
Scoring system
Degree of expansion and hatching status:
1 Early blastocysts
the blastocoel being less than half the volume of the embryo.
2 Blastocyst

the blastocoel being greater than or equal to half of the

volume of the embryo.
3 Full blastocyst
the blastocoel completely fills the embryo.
4 Expanded blastocyst

the blastocoel volume is now larger than that of the early

embryo and the zona is thinning.
5 Hatching blastocyst
the trophectoderm has started to herniate through the zona.
6 Hatched blastocyst
the blastocyst has completely escaped from the zona.
Inner cell mass (ICM) Grading:

A Tightly packed, many cells
B Loosely grouped, several cells
C Very few cells
Trophectoderm Grading:
A Many cells forming a cohesive epithelium
B Few cells forming a loose epithelium.
C Very few cells
30
IVF-ET
Example of a 4AA blastocyst.

Focus on: Inner cell mass
Focus on: Trophectoderm
Table for assessment of blastocysts

Degree of
Inner cell mass
expansion/
hatching
Tightly
Loosely
Very few
status
packed,
grouped,
cells

many cells
several cells

Trophectoderm grading
Many cells
forming a
cohesive
epithelium
Few cells
Very few
forming a
cells
loose
epithelium
A B C A B C

3 Full blastocyst;
the blastocoel
completely fills
the embryo

4 Expanded
blastocyst; the
blastocoel volume
is now larger than
that of the early
embryo and the
zona is thinning

5 Hatching
blastocyst; the
trophectoderm
has started to
herniated through
the zona

6 Hatched
blastocyst; the
blastocyst has
completely
escaped from
the zona
IVF-ET
31
Blastocyst schedule (an example)

The blastocyst culture schedule is quite different from the routine IVF day 2 / day 3
schedule. Here is a day-to-day summary.
We suggest that you make this page visible to all laboratory personnel.
If PLUS products are used no supplementation will be needed. Otherwise, supplement
all media with either G-MM or HSA-solution, except EmbryoGlue, G-GAMETE and
G-RINSE.
Day 1
Day 0
Day 1
(the day before

oocyte collection)
Oocyte collection and

insemination in G-IVF. ICSI
oocytes should be placed
in G-1 immediately after
injection. Prepare G-1
dishes late in the afternoon
for use Day1. Prepare
G-MOPS dishes for oocyte
retrieval.
Assess IVF and ICSI

fertilisation. Rinse in
G-MOPS/G-GAMETE
and place into G-1 for
culture.
Day 2
Day 3
Day 4
Embryo assessment optional.
Assess embryos for cleavage.

Rinse embryos in G-MOPS/
G-GAMETE and wash
well and transfer to G-2 for
culture, early afternoon.
Embryo assessment
optional. Prepare dishes with
EmbryoGlue or G-2 for
transfer and G-2 dishes for
freezing and hold to day 6,
late in the afternoon.
Day 5
Day 6
Assess morphology of
blastocysts. Use scoring
criteria for transfer, freezing or
hold. Transfer blastocysts to
EmbryoGlue or fresh G-2
before transfer to the uterus.
Assess morphology of
blastocysts. Use scoring
criteria for transfer or freezing.
Blastocysts scoring less than
3BB or below by day 6, do not
cryopreserve very well. Transfer
blastocysts to EmbryoGlue
or fresh G-2 before transfer
to the uterus.
Prepare G-RINSE,
G-MOPS and G-IVF
for oocyte collection and
insemination as for routine
IVF protocol. For ICSI
prepare also G-1 dishes.
If G-GAMETE is used,
prepare dishes for oocyte
wash.
32
IVF-ET
Embryo transfer
Embryo transfer (ET) is the process of placing the embryos into the uterus. It is usually performed transcervically, without anaesthesia.
Essentials
Sterile non-toxic non-powdered gloves should be worn when handling the catheter.
Great care should be taken to avoid contamination of the catheter tip at all times.
It is common practice to transfer one or two embryos per patient.
A large volume (60 microliters) of transfer media and a large air interface may result
in expulsion of embryos into the cervix or to the outside of the catheter. Removing
the air column can minimise such complications. Therefore it is recommended that a
continuous fluid column of approximately 30 microliters of equilibrated EmbryoGlue
or equilibrated supplemented G-2/G-2 PLUS is used in a catheter attached to a 1cc
airtight syringe. Load the embryos preferentially toward the tip of the column of transfer
media, closest to the catheter opening.
Replacement of embryos
Hands on for the gynaecologist
1 Arrange in advance for the patient to arrive for the ET with a partially full bladder. In most
cases this helps to position the uterus for easy entry with a soft, atraumatic catheter. In
the situation of a retroverted uterus, the patient may empty her bladder.
2 A non-toxic, quality tested, single use, atraumatic catheter must be used.

3 It is recommended to perform embryo transfer under ultrasound guidance.
4 Place the patient into lithotomy position. Expose the cervix with the use of a speculum
washed with equilibrated G-RINSE with added antibiotics. It may be necessary to
remove the cervical mucus with a sterile syringe.
5 An ultrasound scan during the ET (abdominally) is highly recommended for guidance of

the catheter in its passage through the cervical canal. A trial catheter may be introduced
to test the ease of passage. If the passage is easy, the embryos may be loaded into a
clean catheter and the ET performed. If the trial catheter meets some obstruction several options may be considered. An obturator with some flexibility may be used to direct
the catheter into the cavity. Whichever is used, attention should be paid to minimise
trauma to the cervical canal and cavity.
IVF-ET
33
6 The operator performing the ET should be careful not to expel the embryos from the
catheter by bending, squeezing or causing damage to it.
7 The catheter should pass through the cervical canal and beyond the inner os. Tactile
feedback may be sufficient for the operator to accomplish this. The catheter should be
clearly marked so that it can be determined how far it has been introduced. Ultrasound
should be used to confirm the position.
8 Expel the embryo(s) into the uterus in approximately 30 L of transfer medium and
slowly withdraw the catheter while maintaining steady pressure on the plunger of the
syringe.
9 Make a final microscopic examination of the catheter.
For a more complete review of this topic see: Schoolcraft, Surrey and Gardner
(2001) Embryo transfer: techniques and variables affecting success.
Fertil. Steril.76; 863-870.
34
IVF-ET
Loading the catheter

Hands on for the laboratory
1 Embryo transfer can be performed either in equilibrated EmbryoGlue or in equilibrated
supplemented G-2/G-2 PLUS.
2 Add approximately 1 mL of EmbryoGlue to the well of a rinsed centre well dish.

3 Add approximately 2 mL of EmbryoGlue to the moat of the centre well dish.
4 Pre-equilibrate the dish in +37C and 6 % CO2 for 418 hours.
5 Pre-equilibrate the embryos to be transferred in the well containing EmbryoGlue for
a minimum of 10 minutes in a 6% CO2 environment prior to transfer. Embryos can
be held in EmbryoGlue for up to 4 hours. Embryos should not be kept in EmbryoGlue
overnight.
6 Rinse the 1 mL non-toxic syringe by drawing up and then out media from the moat
several times until no air bubbles are observed in the syringe. Draw up approximately
0.5 mL of the medium from the moat.
7 Firmly attach the transfer catheter to the pre-rinsed 1 mL non-toxic syringe. Flush
approximately 0.51.0 mL of equilibrated transfer medium from the moat through and
out of the catheter.
8 After rinsing, draw approximately 0.1 mL of EmbryoGlue from the center well and expel
into the moat until approximately 20 L is left in the syringe.
9 Under microscopic control, gently load the embryos into the catheter in approximately
510 L of additional EmbryoGlue followed by a small amount of air. (The small pocket
of air at the tip allows better imaging of the tip for ultrasound guided transfers). For the
embryo transfer, pass the tip of the catheter into the uterus approximately 1 cm from the
top of the cavity and expel the embryos in a total volume of approximately 2530 L of
medium. Slowly withdraw the catheter while maintaining steady pressure on the plunger
of the syringe. Make a final microscopic examination of the catheter.
IVF-ET
35
This page is intentionally left blank.
36
IVF-ET
Micro
Manipulation
Micromanipulation includes the procedures of ICSI (Intra Cytoplasmic Sperm Injection),
assisted hatching and embryo biopsy.
Essentials
All micromanipulation equipment should be correctly aligned and positioned for
maximum stability. It is recommended to keep it separate from vibrations caused by
doors, elevators, air currents and through traffic that may cause stressful interruption
and noise.
The inverted microscope should have a heated stage correctly adjusted to maintain fluid
in the dish at +37C. Temperature maintenance during the procedures is important.
Osmolality is controlled by using droplets under equilibrated oil.
Highest quality non-toxic micro-tools should be used for all procedures for consistency
and quality control, see Swemed Instruments by Vitrolife page 62, ICSI- and Holding
Pipettes.
Micro Manipulation
37
Denudation of oocytes for ICSI

If ICSI is to be performed, the oocytes will need to have their cumulus mass and corona
DENS
A GIII
removed.
This
process is called denudation.
This process may be performed either using the large volume method without oil, using
A
multi-wells and dishes or the droplet method under oil.
HYASE (hyaluronidase) is used to facilitate the dispersal of the cumulus mass and corona.
a) glass pipettes, see Swemed Instruments by Vitrolife page 62, Denudation
Using fine
b)cells are removed by gently pipetting the oocyte up and down. The diameter of the
pipettes,
pipette c)
should be slightly larger than that of the oocyte (approximately 130175 m). When
using glass pipettes, several different sized pipettes may be needed. Using a pipette that is
too narrow or has a jagged edge may damage the oocyte. Exposure to HYASE for too long
or rough handling as well as exposure to sub-physiological pH and temperatures, may also
DENS the
B,C,D
GIII
damage
oocyte.
Oocytes in the germinal vesicle stage are particularly sensitive. For the
above reasons it is important to use the correct concentration of HYASE and to keep to the
recommended exposure time. HYASE is concentrated 10 times and should be diluted 1:10
B
D
C
with supplemented G-MOPS/G-MOPS PLUS or with G-GAMETE.
CAUTION
Exposure to HYASE for longer than 30 seconds may damage the oocyte.
a)
Hands on
PREPCULT A,B
1 Dilute HYASE with supplemented G-MOPS/G-MOPS PLUS or with
G-GAMETE.
a)
A
a)
2 Prepare rinsed denudation dishes with diluted HYASE and supplemented

G-MOPS/G-GAMETE for oocyte wash.
b) diluted HYASE, prepare four washing
b) wells of approximately 1mL or
3 For every well of
6 droplets (50100 L) of supplemented G-MOPS/G-MOPS PLUS/G-GAMETE

under oil (A, B). Equilibrate at +37C at room atmosphere for approximately 15 minutes, if G-MOPS/G-MOPS PLUS is used. If G-GAMETE is used, dishes should be
MICRO A,B
equilibrated in 6% CO2 until correct pH has been attained, preferrably over night. At the
same time prepare ICSI dishes (see below HANDS ON - SETUP).
A
a)
f)
b)
a)
c)
b)
d)
c)
e)
d)
A.BDroplets under oil.
a) HYASE solution b f ) Supplemented G-MOPS/G-MOPS PLUS/G-GAMETE wash droplets
b)
38
a)
Micro Manipulation
d)
c)
b)
a)
e)
d)
c)
B. 5-well dish. With or without oil.

a) HYASE solution
be) Supplemented G-MOPS/G-MOPS PLUS/G-GAMETE, wash droplets
4 Using a large bore pipette, see Swemed Instruments by Vitrolife page 62, Transfer
pipette, place 35 oocytes into the diluted HYASE. Gently pipette the hyaluronidase
and the oocytes. The cumulus cells will start to disperse. It is important not to expose
the oocytes to the hyaluronidase solution for longer than 30 seconds.
5 Move the partly denuded oocytes into the first wash volume and take care to carry over
a minimum amount of hyaluronidase solution. Aspirate each oocyte singly up and down,
using a fine bore denudation pipette, see Swemed Instruments by Vitrolife page 62,
Denudation pipette, to remove the corona. Rinse the oocytes in warmed supplemented
G-MOPS/G-MOPS PLUS or in equilibrated G-GAMETE. Repeat with new dishes
until all oocytes are denuded.
6 Observe the oocytes for maturity by examining the presence (M2) or absence (M1) of
a polar body or presence of a germinal vesicle (GV). Place all mature oocytes (M2) into
the prepared ICSI droplets, if the oocytes are to be injected immediately. If the denuded
oocytes are to be incubated for some time before injection, the oocytes should be
placed in supplemented G-1/G-1 PLUS until the time of injection. Immature oocytes
(M1 and GV) may be placed in culture medium for further incubation and maturation.
Hands on - set up
1 Pre-warm supplemented G-MOPS/G-MOPS PLUS and OVOIL at +37C for
approximately 15 minutes, or equilibrate G-GAMETE at +37C and 6 % CO2 for at
least 4 hours.
2 Remove ICSI (viscous sperm injection solution) from cold storage and equilibrate to a
temperature of +20 5C.
3 Dishes for ICSI should be made quickly. Place a 110 L drop of ICSI in the center of
Micro Manipulation
39
a)
d)
c)
an ICSI-dish and 610 L droplets of supplemented G-MOPS/G-MOPS PLUS/GGAMETE, cover with OVOIL. Do not prepare more than one dish at a time to avoid
evaporation in the droplets. Work rapidly and have all items needed close at hand (A, B).
MICRO
SETUP
Make up two
dishesA,B
per patient.
A
B
a)
a)
1
6

b)
3
b)
A. Option 1
a) Oocyte droplets, 1 to 6
(G-MOPS PLUS/G-GAMETE)
b) ICSI
B. Option 2
a) Oocyte droplets, 1 to 6
(G-MOPS PLUS/G-GAMETE)
b) ICSI
LOADSTRAW CRYO 1

4 Warm the dishes at +37C for at least 15 minutes if G-MOPS PLUS is used.
If G-GAMETE is used, equilibrate the dishes at +37C and 6 % CO2 for at least 4hours.
a)
b)
c)
d)
e)
f)
g) h)
i)
ICSI procedure
a) Syringe b) Plastic tube c) Cotton plug sealing cement d) FS 2, 2cm e) Air bubble 1/4 cm
f) FS 2 + embryos, 23 cm g) Air bubble 1/4 cm h) FS 2, 1 cm i) Seal: plastic plug
ICSI is the procedure of injecting the sperm into the oocyte. ICSI enables pregnancies to
be achieved in couples with severe male factor infertility.
LOADSTRAW CRYO 2
Polyvinylpyrrolidone (PVP) is commonly used in ICSI because of its viscous properties. It

slows the motility of the sperm and makes it easier to catch the sperm, allowing correct
immobilisation of the sperm and crushing of the tail membrane before injection. It also
a)
b)
c)
d)
e)
f)
g) h)
i)
allows for controlled injection of the sperm by slowing down the speed of the injection fluid.
This helps to stabilise the microinjection procedure and minimise the volume of fluid that is
injected into the oocyte. All these factors contribute to success of the procedure.
Essentials
a) Syringe
b) Plastic tube c) Cotton plug sealing cement d) BFS 2, 2cm e) Air bubble 1/4 cm
f) BFS 2 + embryos, 23 cm g) Air bubble 1/4 cm h) BFS 2, 1 cm i) Seal: plastic plug
Immobilise the sperm correctly by swiping just below the neck of the sperm with the
injection pipette (crush tail).
Inject sperm deep (5075 % of the oocyte diameter) into the cytoplasm and ensure the
sperm is not dragged out with the injection pipette.
40
Micro Manipulation
To avoid the expulsion of the sperm into the perivitelline space, be sure the oolemma is
broken by gentle suction of a small amount of cytoplasm.
Minimise the volume of ICSI (PVP) injected into the oocyte.
For ICSI and Holding pipettes, see Swemed Instruments by Vitrolife page 62.
Hands on
1 Place a small volume (12 L) of prepared sperm suspension into the centre of the
ICSI droplet. Warm the dishes for a few minutes on a heated stage to allow migration
of sperm to the outer perimeter of the droplet.
2 Prime the injection pipette with ICSI to reduce the risk of the sperm sticking to the
inside of the pipette.
3 Place denuded oocytes into droplets of supplemented supplemented G-MOPS/GMOPS PLUS/G-GAMETE, one oocyte per droplet, maximum 4 oocytes at a time.
4 Immobilise individual sperm by using the injection pipette to crush the membrane
of the sperm tail. It is important not to damage the neck region of the sperm since it
contains the centriole, which is crucial for the migration of chromosomes at cell division.
Inefficient tail crushing will lead to reduced fertilisation rates. Aspirate the individual
immobilised sperm.
5 Move the oocyte droplet into the field of view. Use a holding pipette to secure the
oocyte for injection. Position the oocyte with the polar body at the 7 or 1 oclock position. For minimal risk of damaging the meiotic spindle, inject the sperm at the 4oclock
position. Inject the sperm with the least amount of ICSI possible and after injection,
make sure to stop the flow in the injection pipette. Pay attention to the time the dishes
are outside of the incubator. Fewer oocytes per dish may be set up when the operator is
inexperienced or the case is difficult.
6 After all oocytes are injected, rinse through several drops of supplemented G-1/
G-1 PLUS and then place into prepared G-1 culture systems for overnight culture.
7 The oocytes are assessed for fertilisation by the presence of two PN and two polar
bodies on the following day. Non-fertilised, degenerated oocytes and oocytes with
more than 2 PN or less than 2 PN should be removed from culture. After assessment,
the zygotes should be rinsed in equilibrated supplemented G-1/G-1 PLUS and then
transferred to a new dish with equilibrated supplemented G-1/G-1 PLUS for culture.
Micro Manipulation
41
Difficult ICSI cases

The level of difficulty of a case is mainly determined by three factors:
Operator
Equipment
Sperm sample
The training, experience, endurance, and ability of the operator determine the first factor. It is very
important when setting up ICSI that sufficient time and resources are available, particularly for difficult cases. The equipment should be easy to use and provide a stable setting to enable the operator
to concentrate on the difficulties encountered in trying to find and pick the sperm. An ergonomically
designed ICSI workstation allows the work to progress smoother and increases the endurance of
the operator.
The sperm sample can also present difficulties. For example, sperm suspensions prepared from testicular biopsies as well as severe cases of oligoasthenoteratospermia usually exhibit very low count,
low and sluggish motility, and presence of debris and other cells. Frozen thawed epididymal sperm
may also be problematic. Only a small fraction of the sperm may survive and the surviving sperm may
exhibit a very low progressive movement or no movement at all.
A third example of a difficult ICSI case is sperm obtained by Electro-ejaculation. In this case, the
sperm count can be very high and the motility very low. In addition there are other cells and debris
present.
Low sperm count, motility and progression

Use SpermGrad to retrieve sperm (see Sperm Preparation). Add the sperm suspension to
a droplet of equilibrated supplemented G-IVF/G-IVF PLUS. Transfer the sperm to ICSI
before proceeding.
Note that in cases of extremely low sperm count, the semen sample can be washed in
equilibrated supplemented G-IVF/G-IVF PLUS and centrifuged at 300600g for 10
minutes without previous gradient centrifugation. Re-suspend the spermatozoa in
200500 L of equilibrated supplemented G-IVF/G-IVF PLUS and use as soon as
possible.
42
Micro Manipulation
Testicular biopsy
Use G-MOPS PLUS to collect the sample. Transfer the tissue to a sterile non toxic Petri
dish and disperse the material into small pieces.
Transfer the dispersed material to small conical test tubes and centrifuge for 5 minutes
at 300-600g. Discard the supernatants and re-suspend the pellets in G-MOPS PLUS.
Asecond wash may be necessary.
Place 5-10L droplets of the testicular material in a small Petri dish and cover with OVOIL.
Retrieve the testicular spermatozoa and collect in a separate clean droplet of
G-MOPS PLUS until injection.
If the preparations are to be used the following day, the material should be incubated in supplemented G-IVF/G-IVF PLUS at +37C and 6% CO2.
Embryo biopsy
Embryo biopsy is the procedure of removing a blastomere from the embryo for
Pre-implantation Genetic Diagnosis (PGD).
Essentials
Embryo biopsy is usually performed on the morning of day 3, before compaction of the
blastomeres begins.
A hole in the zona can be drilled either with Tyrodes acid, a laser or by partial zona
dissection.
A separate pipette is needed to remove the biopsied blastomere. See Swemed
Instruments by Vitrolife page 62, Blastomere Biopsy Pipette.
CAUTION
G-PGD is MOPS buffered and must be handled exactly like G-MOPS (see
page8). G-PGD must never be placed in a CO2 incubator, but rather warmed in
a tube warming block or in an incubator without CO2.
Micro Manipulation
43
Hands on
1 Prepare dishes with droplets of pre-warmed supplemented G-PGD covered with
OVOIL.
2 Wash the embryo in several droplets of supplemented G-PGD to remove the culture
medium.
3 Place the embryo in G-PGD (MOPS buffered, Ca2+- and Mg2+-free medium) at +37C.
4 Secure the embryo with the holding pipette.
5 Drill a hole in the zona. Introduce a clean biopsy pipette into the hole and gently aspirate
one or two blastomeres for analysis.
6 After extensive washing in G-2/G-2 PLUS, to remove G-PGD, place the embryos
into equilibrated supplemented G-2/G-2 PLUS for culture until the results of the
embryo biopsy analysis are confirmed. The non-affected embryos may be selected for
fresh embryo transfer on day 5, or cryopreservation if there is a surplus. The embryo
transfer is usually performed on day 5, after the genetic diagnosis has been completed.
CAUTION
Spend time adjusting the embryo position on the holding pipette so that a
nucleated blastomere can be clearly identified and biopsied.
If compaction has occurred, there will be junctions between the blastomeres
and they may be more difficult to remove. Be patient, pull and move the
pipette across and back to ease the blastomere out.
Jagged edges on the biopsy pipette will cause the biopsied cell to lyse.
Therefore, only use the highest quality pipettes.
44
Embryo
Cryopreservation
Essentials
The results of cryopreservation are dependent on the baseline IVF success rate of the
clinic. If the fresh IVF results are low, this will also be reflected in the cryopreservation
results.
The results are dependent on well maintained and accurately calibrated freezing
equipment, careful handling and detailed attention to choosing only embryos with good
morphological appearance.
Equipment list
This is a suggested list and alternatives may exist.
1 Cryopreservation machine: Base your purchase decision on cost, available space and
ability for maintenance and service.
2 Storage vessel: This should be a vessel used only for human embryos. Liquid nitrogen
submersion is the traditional method. Separate vessels should be used if you cryopreserve embryos from patients with infectious diseases.
3 Straws or plastic cryo-vials for freezing: These need to be sterile, non-toxic and of high
quality.
4 The amount of liquid nitrogen (LN2) used is dependent upon the choice of equipment
and storage system.
45
Cryopreservation is the freezing and thawing of cells with minimal damage. This is achieved by the
use of a cryoprotectant that dehydrates the cells and disturbs crystallisation of water during cooling.
Selecting an appropriate temperature profile during cooling further facilitates this process so that
the cells become maximally dehydrated. Introduction of cryoprotectant is done in a fashion that is
designed to reduce the risk of osmotic rupture and toxic effects on the cells of high osmolality and
high concentrations of cryoprotectant.
The choice of cryoprotectants used depends on the cell development of the embryos. Propanediol
is commonly used for cleavage stage embryos and glycerol is commonly used for the blastocyst
stage of development. It is important to know that cell damage does not occur in the storage phase
if stored in the liquid phase of liquid nitrogen. It is the freezing and thawing process that is crucial to
cell survival.
The embryos are placed into cryoprotectant solutions and then into containers such as straws or
vials. A biological freezer then lowers the temperature around these containers at an accurately
controlled rate. At the freezing point of the solution, ice crystal formation is induced by touching the
containers with liquid nitrogen dipped forceps. This process is called seeding or ice nucleation. If
this process is forgotten or inadequately performed the cells will suffer damage due to insufficient
dehydration and intracellular ice formation. The formation of extra-cellular ice is a crucial event for the
start of controlled water loss from the embryo.
As the external concentration of the non-permeating solutes increases and the water freezes outside
the embryo, water flows out, thereby dehydrating the embryo. This process continues until the
embryo reaches its freezing point. If the cooling is too fast the embryo will not become sufficiently
dehydrated.
If the embryo is thawed too slowly after freezing the super-cooled water will crystallise during the
slow warming and intracellular ice will cause damage. The rate of thaw is usually related to the cryoprotectant used and the rate of cooling beyond 40C. If the embryo freeze is a fast freeze, i.e. fast
cooling rate to 40C, the thaw rate will be fast i.e. plunging into +30C water bath (500C/min).
Alternatively if the freeze is a slow freeze, i.e. slow cooling rate to 80C the thaw must be performed
in the freezing chamber at around +10C /minute. Glycerol requires a rapid thaw.
More information about the Cryo protocols can be found in:
Edgar DH, Karani J, Gook DA. Increasing dehydration of human cleavage-stage

embryos prior to slow cooling significantly increases cryosurvival. Reprod Biomed
Online. 2009 Oct;19(4):521-5
Gardner DK, Maybach J, Lane M (2001) Hyaluronan and RHSA increase blastocyst
cryosurvival. Proc 17th World Congress on Fertility and Sterility, Melbourne. pp 226
Lane et al (2003) Cryo-survival and development of bovine blastocysts are enhanced by
culture with recombinant albumin and hyaluronan. Mol Reprod Dev 64:7078
Gardner DK et al (2003) Changing the start temperature and cooling rate in a slowfreezing protocol increases human blastocyst viability. Fertil and Steril 79 (2): 407410
46
b)
b)
Cryopreservation of cleavage stage embryos using

MICRO A,B Cleave and Thawkit Cleave
Freezekit
This Amethod of cryopreservation can be used for pronuclear stage oocytes up to the 8-cell
a)
stage. f)
b)
a)
b)
Freezing procedure
c)
d)
FreezeKit Cleave contains two

solutions for freezing of pronuclear-stage oocytes and
c)
e)
cleavage-stage embryos. The solutions consist of MOPS buffered solution and human
d)
serum albumin.
B
Equilibration solution, ES, contains no cryoprotectants

b)
a)
Freezing solution, FS, contains 1,2 - propanediol and sucrose as cryoprotectants for
dehydration of cells.
d)
c)
Essentials
Only cryopreserve embryos of high quality since the thaw survival rate is related to
initial quality of the embryo.
Check
patients
identity
MICRO
SETUP
A,B and prepare all paperwork before you start the procedure.
Hands
A on
a)
1
1 Label dishes and
straws, pipette appropriate volumes (0.5-1.0 ml) of ES and FS into
a)
6
respective
dishes.
b)
2 Equilibrate to room temperature. To avoid dilution, always transfer the embryos in a

3
5
minimum
amount of solution.
b)
3 Place the embryos selected for freezing in ES and rinse properly. The embryos can stay
in ES for 10 minutes.
4 Transfer
the embryos
into the
LOADSTRAW
CRYO
1 FS and expose for 10 minutes. Loading of the prepared
straws can start immediately after the embryos have been transferred to FS. The total
exposure time for the embryos to the FS should be 10 minutes. Load the straws in
accordance with manufacturers recommendation or as shown below.
a)
b)
c)
d)
e)
f)
g) h)
i)
a) Syringe
f) FS + embryos, 23 cm
a)Plastic
Syringe
b) Plastic
tube c) Cotton
plug
sealing
cement d) FS 2, 2cm e) Air bubble 1/4 cm
b)
tube

g) Air
bubble
1/4 cm
c) Cotton plug sealing cement
h) FS, 1 cm
d) FS, 2cm
i) Seal: plastic plug
e) Air bubble 1/4 cm
LOADSTRAW CRYO 2
a)
47
b)
c)
d)
e)
f)
g) h)
i)
5 Place the straws in the freezing machine at room temperature and initiate the freezing
program.
Use the following freezing program

Starting temperature
+18.0 to +25C
Step 1
2.0C/min to 6.0C
Step 2

Hold at 6.0C and manually seed after 2 minutes. Keep the

straw at 6.0C for a total time of 10 minutes.
Do not seed close to the embryo
Step 3
0.3C/min to 30C
Step 4
50C/min to 150C
Plunge the straw into liquid nitrogen and arrange for storage at -196C.
Store submerged in LN2 (not in the vapor phase)
Thawing procedure
ThawKit Cleave contains three solutions for thawing of pronuclear-stage oocytes and
cleavage-stage embryos. The solutions consist of MOPS buffered solution and human
serum albumin.
Thawing solution 1, TS1, and Thawing solution 2, TS2, are buffered solutions containing
decreasing concentrations of sucrose allowing removal of 1,2 -propanediol and gradual
rehydration of the cells.
Equilibration solution, ES, is a buffered solution that does not contain any cryoprotectants
and is used for final rehydration before culture.
Essentials
48
Thaw straws one at a time.
Perform all steps at ambient temperature.
Check patient identity carefully and prepare all paperwork accordingly.
Hands on
1 Label dishes, pipette appropriate volumes (0.5-1.0 ml) of TS1, TS2 and ES into
respective dishes. Equilibrate to room temperature. To avoid dilution, always transfer
embryos in a minimum amount of solution.
2 Remove the straw from liquid nitrogen and expose to air for 30 seconds.
3 Place the straw in a 30C water bath for 45 seconds.
4 Remove the straw and wipe it carefully. Open the straw using aseptic technique and
gently expel the embryos. Expose the embryos to TS1 for 5 minutes.
5 Move the embryos into TS2 and expose for 5 minutes.
6 Move the embryos into ES and expose for 5 minutes.
7 Transfer the embryos to accurately equilibrated culture medium, rinse embryos properly
and culture according to standard laboratory procedure.
Cryopreservation of cleavage stage embryos using

Freezekit 1 and Thawkit 1
This method of cryopreservation can be used for oocytes, pronuclear stage oocytes up to
8-cell embryos.
The method is adapted from the original method of Testart et al (1986), using 1,2 propanediol
(PrOH) as a permeating cryoprotectant. Sucrose is also used as a non-permeating cryoprotectant.
Sucrose is a large molecule that osmotically promotes dehydration during cooling and protects
against cell lysis when thawing the embryos. A phosphate buffered solution is used so that all steps
may be performed outside the incubator and at ambient temperature.
FREEZE-KIT1
Cryo-PBS
= Phosphate buffer solution with 25mg/mL Human serum albumin (HSA)
Freeze solution 1 = FS1
= 1.5M PrOH in Cryo-PBS
Freeze solution 2 = FS2
=1.5M PrOH + 0.1 M sucrose in Cryo-PBS
49
b)
b)
MICRO A,B
Example of freezing program for

cleavage
stage embryos
A
a)
f)
b)
a) cryoprotectant equilibration
b)
The duration of the freezing procedure including
is approximately 2 h.
c)
Starting
e) temperature
c) +18.0 to +25C
d)
Step 1
2.0C/min to 7.0C
Step 2
d)
b)
HOLD AT 7.0C for 10 min, SEED after 2 min
Stepa)3
0.3C/min to 30.0C
Step 4
30.0C to below 80.0C (at least 10C/min)

d)
c)
Remove straws and plunge into LN2, store submerged in LN2 (not in the vapor phase).
Essentials
Only cryopreserve embryos of high quality since the thaw survival rate is related to initial
MICRO SETUP A,B
quality of the embryo.
Check patients identity and prepare all paperwork before you start the procedure.
A
Hands on
6
a)
a)
5
2
b) pipette appropriate
1 Label dishes for freezing solutions,
volumes of Cryo-PBS,
FS1, and
3
FS2 into
respective dishes.
Equilibrate to ambient temperature.
3
5
b)
2 Observe and record detailed morphology. Rinse embryos for freezing in Cryo-PBS.
3 Gently place embryos into FS1 for 10 minutes (never more than 20 minutes). The cells
of the embryo will shrink and then re-equilibrate during this step.
LOADSTRAW CRYO 1
4 Move embryos across to FS2 and then load into straws by attaching the straw to a 1 mL
syringe (syringes are connected to the straws by using 1 cm of silastic tubing). Remove
the straws and seal, so that liquid nitrogen will not leak inside the straw.
a)
b)
c)
d)
e)
f)
g) h)
i)
a) Syringe
f) FS 2 + embryos, 23 cm
b)
Plastic
tube
Air bubble
1/4cement
cm
a) Syringe b) Plastic tube c) Cottong)plug
sealing
d) FS 2, 2cm e) Air bubble 1/4 cm
c)f) Cotton
plug sealing
cement
h) FS
2, cm
1 cm
FS 2 + embryos,
23
cm g) Air bubble
1/4
h) FS 2, 1 cm i) Seal: plastic plug
d) FS 2, 2cm
LOADSTRAW CRYO 2
50
a)
b)
c)
d)
e)
f)

g) h)
i)
5 Place into freezing chamber at ambient temperature and commence freezing program.
6 Manually seed the straws at 7C with liquid nitrogen (LN2) cooled forceps close to the
cotton plug.
Do not seed the straw close to the embryos. Do not drop straw or shake it. If there are air
bubbles in the straw it may reduce cell survival. Continue the freezing program.
7 The freezing takes approximately 2 hours. Attention must be paid to the handling of the
straws at low temperatures as they may break very quickly. Plunge into liquid nitrogen
and arrange for storage at 196C.
Thawing program for cleavage stage embryos

THAW-KIT 1
Thaw solution 1 = TS1
= 1.0 M PrOH + 0.2 M Sucrose in Cryo-PBS
= 0.5 M PrOH + 0.2 M Sucrose in Cryo-PBS
= 0.2 M Sucrose in Cryo-PBS
Essentials
Thaw straws one at a time.
Perform all steps at ambient temperature.
Check patient identity carefully and prepare all paperwork accordingly.
Hands on
1 Identify patient and location of straw. Prepare all paperwork. Keep the straw under LN2
in small Dewar vessel until actual thawing. Label dishes for thaw solutions and pipette
appropriate amounts of TS1, TS2, TS3 and Cryo-PBS.
2 Remove straw and air thaw for 30 seconds. During this time handle the straws carefully
and examine it for air bubbles, cracks in the seal and any leakage of LN2.
3 Place in a +30C water bath for 30 seconds. Remove and carefully wipe. Cut the plug
end of the straw with sterile scissors, and attach it to a 1 mL syringe. Cut the other end
carefully, do not shake straw or make air bubbles.
51
4 Gently expel the embryos into TS1. Observe the embryos coming out of the straw and
if you do not see them all, quickly refill the straw and gently flush. Occasionally the
embryos will stick to the sides of the straw.
5 Incubate the embryos for 5 minutes in TS1.

6 Gently move the embryos to TS2 for 5 minutes (approximately). Remember that these
embryos are osmotically STRESSED and need to be handled VERY CAREFULLY.
7 Move the embryos to TS3 for 510 minutes. The embryos will be stressed and the membranes will be very fragile.
8 Place embryos in Cryo-PBS at ambient temperature for 6 minutes, followed by 4 minutes at 37C on a heated stage. Do not place in the CO2 incubator, as the buffer capacity of Cryo-PBS is insufficient for the 5 % CO2 atmosphere.
9 Embryos may be placed into equilibrated supplemented G-1/G-1 PLUS (or

G-2/G-2 PLUS if the embryos were cryopreserved on day 3). Assess and record survival and morphology compared to pre-freeze. Embryos may be transferred immediately
or left for further culture. Pronuclear oocytes should be cultured overnight in G-1/G-1
PLUS and only transferred if normal cleavage has occurred.
10 The thaw-cycle is very important to the success of embryo cryopreservation. Ensure that
the embryo transfer is performed on the appropriate day. Embryos may be replaced in a
natural or artificial cycle.
Cryopreservation of blastocyst stage embryos

G-FreezeKit Blast
Supplement with either 50 mg/mL G-MM or 100 mg/mL HSA-solution:
Blastocyst incubation medium (BIM)
= 9.0 mL BIM + 1.0 mL G-MM or 1.0 mL HSA-solution.
Blastocyst freezing solution 1 (BFS 1)
= 9.0 mL BFS 1 + 1.0 mL G-MM or 1.0 mL HSA-solution.
Blastocyst freezing solution 2 (BFS 2)
= 9.0 mL BFS 2 + 1.0 mL G-MM or 1.0 mL HSA-solution.
BIM is based upon a modified MOPS-buffered G-2 medium without sucrose and glycerol.
BFS1 contains 100 mM of sucrose and 5 % glycerol
BFS2 contains 200 mM of sucrose and 10 % glycerol
52
Example of freezing program for blastocyst stage

embryos
Starting temperature 6C
Step 1
Seed after 2 minutes
Step 2
HOLD for 10 minutes
Step 3
Cool at 0.5C/min to 32C
Remove straws and plunge into LN2 , store submerged in LN (not in the vapor).
2
Essentials
Only cryopreserve blastocysts of high quality ( 3BB) since the thaw survival rate is
related to the initial quality of the embryo.
Check patient identity and prepare all paperwork before the procedure is started.
Hands on
1 Label dishes for BIM, BFS 1 and BFS 2. Pipette 800 L volumes of BIM, BFS 1 and
BFS 2 into respective dishes. Equilibrate to +20 5C.
2 Observe and record detailed morphology. Rinse blastocysts for freezing in BIM.
3 Gently place embryos into BFS 1 for 10 minutes.
4 Move embryos across to BFS 2 for 7 minutes.
5 Use minimal volumes when moving embryos.
6 During this 7 minutes rinse the freezing straw with BFS 2 in a separate dish/well.
7 After 7 minutes, transfer the blastocysts to a new well with BFS 2 for loading into
straws.
53
a) Syringe b) Plastic tube c) Cotton plug sealing cement d) FS 2, 2cm e) Air bubble 1/4 cm
8 Load the blastocysts into pre-rinsed straws by attaching the straw to a 1 mL syringe
(syringes can be connected to the straws by using 1 cm of silastic tubing). Remove the
LOADSTRAW CRYO 2
straws and seal, so that liquid nitrogen will not leak inside the straw.
Do not exceed 15 minutes in BFS 2.
a)
b)
c)
d)
e)
f)
g) h)
i)
a) Syringe
f) BFS 2 + embryos, 23 cm
b)a)Plastic
tube
Air bubble
1/4cement
cm
Syringe b) Plastic tube c) Cottong)plug
sealing
d) BFS 2, 2cm e) Air bubble 1/4 cm
c)f)Cotton
sealing 23
cement
h) BFS
2, 1
cm
BFS 2 +plug
embryos,
cm g) Air bubble
1/4
cm
h) BFS 2, 1 cm i) Seal: plastic plug
d) BFS 2, 2cm
9 Immediately place into freezing chamber at 6C.

10 Wait 2 minutes.
11 Manually seed the straws at 6C with LN2 cooled forceps at the top of the column
containing the embryos. Do not seed the straw close to the embryos. Do not drop straw
or shake it. If there are air bubbles in the straw, cell survival may be reduced. Hold the
temperature for a further 10 minutes and then initiate the freezing program.
12 The freezing takes approximately 50 minutes. Attention must be paid to the handling of
the straws at low temperatures as they may thaw very quickly. Plunge into liquid nitrogen
and arrange for storage at 196C.
Thawing Program for blastocyst stage embryos

G-ThawKit Blast
Supplement with either 50 mg/mL G-MM or 100 mg/mL HSA-solution:
Blastocyst thaw solution 1 (BTS 1)
= 9.0 mL of BTS 1 + 1.0 mL G-MM or 1.0 mL HSA-solution
Blastocyst incubation medium (BIM)
= 9.0 mL of BIM + 1.0 mL G-MM or 1.0 mL HSA-solution
54
BTS 1 contains 200 mM of sucrose and 10 % glycerol

BTS 2 contains 100 mM of sucrose and 5 % glycerol
BTS 3 contains 100 mM of sucrose.
BIM is based upon a modified MOPS-buffered G-2 medium without sucrose and glycerol.
Hands on
All procedures take place at room temperature.
1 Label dishes for BIM, BTS 1, BTS 2, and BTS 3. Pipette 800 L volumes of BIM, BTS
1, BTS 2 and BTS 3 into respective dishes or wells. Equilibrate to +20 5C.
2 Fill cryo-dewer with LN2 and quickly remove canes from cryo-storage tanks and place
into LN2 dewer.
3 Remove straw from cane using forceps and thaw in air for 10 seconds.
4 Place straw in a 30C water bath for 30 seconds.
5 Remove straw from water bath and dry thoroughly with a wipe. Cut the cotton end of
the straw and insert cut end into a 1 mL syringe. The syringe will be used with the straw
inserted into it to expel the embryos.
6 Cut the other end of the straw and insert cut end into BTS 1. While looking through the
microscope, expel embryos into BTS 1 using the syringe. Expel any additional liquid on
to the dish cover and save to search through if all embryos are not recovered in BTS 1.
The time taken from removing the straw from the waterbath to expelling into BTS 1
should be minimal (less than 1 minute). Only one straw should be thawed at a time.
7 Using a pulled pipette that is just slightly bigger than the embryos, move the embryos
immediately into BTS 2 for 5 minutes. Transfer as little media as possible.
8 Transfer embryos to BTS 3 for 5 minutes.

9 Transfer embryos to BIM for 5 minutes.
10 Transfer embryos to a second dish of BIM warmed on a stage warmer at +37C for
5minutes.
11 Wash embryos in pre-equilibrated supplemented G-2/G-2 PLUS once in the well of a

1-well dish and place embryos into culture.
12 Embryos should be transferred to the uterus in equilibrated EmbryoGlue or in equili
brated supplemented G-2/G-2 PLUS.
55
Quality
Control Program
All raw materials used for manufacturing Vitrolife products are dedicated for human
medical use and whenever applicable are of US Pharmacopeia (USP) and European
Pharmacopoeia (Ph Eur) grade. Each lot of raw materials and final products are tested and
evaluated by stringent quality control procedures. All production takes place in a manufacturing environment classified for aseptic production that is under strict control and monitoring. Quality Control, together with quality assured operations (ISO 13485:2003 and 21
CFR Part 820:QSR) result in excellent LOT-to-LOT consistency.
Physicochemical, biological and functional tests are performed according to the characteristics of each product. In addition to recognised standards for test performance, internal
Standard Operating Procedures according to the Quality Handbook of Vitrolife are followed. The Quality Control Program of Vitrolife includes all necessary tests to guarantee
both the safety and efficacy of the media.
All instruments and standard solutions used in the Quality Control system are acquired from
qualified manufacturers.
Validation and calibration

All quality control procedures performed by Vitrolife are performed in accordance with
quality assured operations (ISO 13485:2003 and 21 CFR Part 820:QSR) by highly
qualified personnel. All methods and equipment used are subject to a continuous and
extensive validation and calibration program.
56
Physicochemical tests
pH
The pH measurement is performed using a validated method according to USP and Ph
Eur. The range of acceptance for each product is kept as small as possible, considering the
nature of the product and its intended use. Samples are pre-incubated at the appropriate
temperature and atmosphere before measurement. Media equilibration at 6 % CO2 is
achieved by the use of a gas mixture with a certified CO2 level.
Osmolality
The osmolality measurement is performed according to USP and Ph Eur using a validated
method based on freezing-point depression. The range of acceptance for each product is
kept as small as possible, considering the nature of the product and its intended use.
Biological tests
Bacterial endotoxin
The absence of toxic levels of endotoxins is verified for all raw materials and each LOT
manufactured. The validated bacterial endotoxin test (LAL assay) that is used by Vitrolife
is the most sensitive of the currently used methods for endotoxin testing with a minimum
sensitivity of 0.005 EU or IU/mL. The validated procedure is performed in accordance with
USP, Ph Eur, and FDA guideline Guideline on validation of the limulus amebocyte lysate
test as an end-product endotoxin test for human and animal parenteral drugs, biological
products, and medical devices.
Sterility test - membrane filtration

The sterility of all media is confirmed by a membrane filtration in accordance with USP and
Ph Eur. The test period for media is 2 weeks and for oil 3 weeks. No detectable bacterial or
fungal growth is accepted, neither in fluid thioglycollate medium nor in soya-bean casein
digest medium. A strict requirement of the sterility assurance level (SAL) provides a safe
application for users.
57
Functional tests
Mouse embryo assay
Mouse embryo assay (MEA) is a functional test method. All media, medium components
and critical devices used for media manufacturing are tested by culturing one-cell stage
mouse embryos to the expanded blastocyst stage. The developmental stages of the
embryos are recorded according to the specifics of each test. The safety and efficacy
of the media is determined by observing the number of embryos that develop to defined
developmental stages with appropriate cell numbers in a predetermined time period. In
addition, the determination of cell numbers and the use of specific cut off values set for all
products increase the sensitivity of the MEA as cell numbers of the blastocysts are linked to
their subsequent viability upon transfer to the uterus.
Validation of Mouse Embryo Assay for Screening Medical Devices in Clinical

ART
A bioassay, such as the one cell Mouse Embryo Assay (MEA), is required for laboratory
certification by the College of American Pathologists which governs Reproductive Clinics
in the United States. All commercial media and those media made in house, must be evaluated prior to clinical use by such an assay.1 The MEA is the most widely used assay for
media components, culture media, and equipment used in clinical ART.2 The MEA has been
proven effective in screening for several potential embryo toxins present in the equipment
used in clinical work, new LOTs of oil used as a media overlay, consumables and each new
LOT of medium. Many steps have been taken to maximise the sensitivity of the MEA. For
example, when testing contact materials we use media without albumin as it can chelate
potential embryo-toxins. Furthermore, we use 1-cell mouse embryos as opposed to 2-cell
stage.3
Determining cell numbers of blastocysts in the MEA is indicative of embryo viability.
As shown in the figure below, fetal development is proportional to blastocyst cell number.4
Using the MEA it is possible to quantitate the product properties of culture media used in
clinical ART.
REFERENCES
1. College of American Pathologists (1998), Reproductive LaboratorySection 90 Proposed Checklist, p26
2. Gardner DK and Lane M (1999), Embryo culture systems. Handbook of
In Vitro Fertilisation (Second Edition), Eds. Trounson AO and Gardner DK,
CRC Press, Boca Raton p 205-264.
3. Davidson, A., Vermesh, M., Lobo, R.A., and Paulson, RJ (1998), Mouse
embryo culture as quality control for human in vitro fertilisation: the one-cell
versus the two-cell model. Fertil. Steril., 49: 516-521.
4. Lane M. and Gardner DK (1997), Differential regulation of mouse embryo
development and viability by amino acids. Reprod Fertil., 109: 153-164.
58
G-Series and related products from Vitrolife

Product
510(k)# Intended purpose
G-MM
K021894

G-MM contains Recombinant Human Albumin solution

(50 mg/mL) and is intended for use in assisted
reproductive procedures which include gamete and
embryo manipulation. These procedures include the use
of G-MM as a supplement for culture medium.
Not for use as an injectable product.
HSA-solution
K021896

HSA-solution contains Human Serum Albumin solution

(100 mg/mL) and is intended for use in assisted
reproductive procedures which include gamete and
embryo manipulation. These procedures include the use
of HSA-solution as a supplement for culture medium.
Not for use as an injectable product.
CAUTION: All blood products should be treated as potentially
infectious. Source material from which this product was derived was
found negative when tested in accordance with current FDA required
tests, HIV, types 1 and 2; HBV; HCV, and HTLV types I and II. No
known test methods can offer assurance that products derived from
human blood will not transmit infectious agents.
G-IVF
K081116
G-IVF PLUS
Medium for preparation and handling of gametes

and for in vitro fertilisation.
G-1
K081114
G-1 PLUS
Medium for culture of embryos from the pronucleate

stage to day 2 or day 3.
G-2
K081117
G-2 PLUS
Medium for culture of embryos from day 3

to the blastocyst stage.
EmbryoGlue
Medium for embryo transfer.
K031015
G-MOPS
K081115
G-MOPS PLUS
Medium for handling and manipulating

oocytes and embryos in ambient atmosphere.
G-GAMETE
K021893

For handling and manipulating oocytes

and embryos in ambient atmosphere.
G-RINSE
K022295

Solution for rinsing of contact materials and for

washing of the cervix. Not for culture.
continued on next page
59
Product
510(k)# Intended purposee
G-FreezeKit Blast K032154
Media for freezing of blastocyst stage embryos.
G-ThawKit Blast K032155
Media for thawing of blastocyst stage embryos.
G-PGD
K033101
Medium for embryo biopsy.
SpermGrad
K023403
For gradient sperm separation.
HYASE *
K000627
For removal of cumulus cells.
ICSI *
K043116

For the immobilization and isolation of sperm prior to

intracytoplasmic sperminjection, ICSI.
FREEZEKIT 1 *
K000623
For freezing of cleavage stage embryos.
THAWKIT 1 *
K000618
For thawing of cleavage stage embryos.
FreezeKit Cleave *

Solutions for freezing of pronuclear oocytes and

cleavage-stage embryos.
ThawKit Cleave *

Solutions for thawing of frozen pronuclear oocytes and

cleavage-stage embryos.
OVOIL

K991351

For covering of medium during in vitro fertilisation and

micro-manipulation procedures.
Additional products supplied by Vitrolife

IVF *
K991348

For in vitro fertilisation, culture and transfer

of embryos.
ASP *
K991345
For oocyte retrieval and rinsing (follicle flushing).
SpermRinse *
K000621
For sperm preparation.
CCM *

For
research
use only
For culture from day 3 to blastocyst stage and transfer.
* Not available in Australia

Not available in the US
60
G-Series: Media Contents

Medium
Antibiotic
MOPS NaHCO3 Amino acids Vitamins Hyaluronan
G-RINSE
Gentamicin
Yes
G-MOPS /
G-MOPS PLUS
Gentamicin
Yes
Yes
Non ess
G-IVF /
G-IVF PLUS
Gentamicin
Yes
Non ess
G-GAMETE
Gentamicin
Yes
Yes
Non ess
G-1 / G-1 PLUS Gentamicin
Yes
Non ess
Yes
G-2 / G-2 PLUS Gentamicin
Yes
Non ess +Ess
Yes
Yes
EmbryoGlue
Gentamicin
Yes
Non ess +Ess
Yes
Yes
G-FreezeKit Blast
Gentamicin
Yes
Yes
Non ess
G-ThawKit Blast
Gentamicin
Yes
Yes
Non ess
FreezeKit Cleave
Gentamicin
Yes
Yes
Non ess
Yes
ThawKit Cleave
Gentamicin
Yes
Yes
Non ess
Yes
G-PGD
Gentamicin
Yes
Yes
Non ess
Media contents G-Series
61
For further information and ordering see www. vitrolife.com
The availability of these Instruments may be subject to National Regulatory Requirements.
Product
62
510(k)# Intended purpose
Follicle Aspiration K991273

Needles, single,
double and
luer lumen needles
with several lengths
and diameters
The Follicle Aspiration Set is a sterile, single use device

which is intended for flushing and /or aspiration of
oocytes from the ovarian follicle
Denudation
K991700
Pipettes, several
diameters
The denudation pipettes are intended for removal of the

cumulus cell layers of the oocyte
Transfer Pipettes, K991700

two diameters
fertilisation
The Transfer pipettes are intended for manipulation and

transfer of oocytes, embryos and blastocysts, or to check
ICSI Pipettes
K991700
w/o spike,
Several diameters,
angles and lengths
The ICSI Pipettes are intended for intra cytoplasmic

single sperm injection of oocytes
ICSI Pipettes
K991104
w spike, Several
diameters,
angles and lengths
The ICSI Pipettes are intended for intra cytoplasmic

single sperm injection of oocytes
Holding Pipettes, K991700

Several diameters, K991104
angles and lengths

The Holding pipettes are intended to hold the oocyte or

embryo in position with the application of vacuum during
intracytoplasmic single sperm injection or other micro
manipulating procedures.
Blastomere Biopsy K022643

Pipette, several
diameters and
angles
The Blastomere biopsy pipettes are intended to conduct

a blastomere biopsy which may be done in order to
perform pre-implantation genetic diagnosis of the genetic
material in the biopsied cell(s).
Product
510(k)# Intended purposee
Partial Zona
K991700 The Partial zona dissection pipettes are intended to make a
Dissection
hole in the zona pellucida to enable embryo assisted
Pipette hatching.
Hatching Pipette, K991700
two diameters
and lengths
Microcell
Sperm counting
chamber

The Assisted hatching pipettes are intended to make a

hole in the zona pellucida to enable embryo assisted
hatching.
---
The Microcells are intended for counting of spermatozoa
Not
Medical
Device
63

Do not use if product appears cloudy.
To preserve sterility, Vitrolife recommends that it be opened and used only with aseptic
technique.
Media bottle should not be stored after opening. Discard excess media after completion of
procedure.
For external use only. Not for use as an injectable product.
Caution: US Federal law restricts this device to sale by or on the order of a physician.
CAUTION: All blood products should be treated as potentially infectious. Source material
from which this product was derived was found negative when tested in accordance with
current FDA required tests, HIV, types 1 and 2; HBV and HCV. No known test methods can
offer assurance that products derived from human blood will not transmit infectious agents.
The risks of reproductive toxicity and developmental toxicity for IVF media, including
G5Series of IVF Media, have not been determined and are uncertain.
Further information
This written material contains limited information regarding Vitrolife products. The
information on various tests and clinical trials herein is only a summary provided for
background information purpose about these products. This information should not be
considered as complete, nor is it a substitute for the advice of a physician. Therefore, it is
important to discuss the included information with a physician. If you are a physician, you
are recommended to revert with Vitrolife or its authorised agents or distributors for complete
information on products and procedures related herein.
Moreover, this written material contains information relating in particular to health, suitability,
the medical domain and various kinds of medical treatment reserved exclusively for use by
human beings.
All products mentioned in this written material may not be available in all countries. For
64
more information contact Vitrolife or the Vitrolife distributor. The recommendations for use
of the products may have changed since the printing of this written material. For the latest
information please consult the Vitrolife homepage (www.vitrolife.com).
Please note that all purchases of Vitrolife media and instruments products from Vitrolife are
subject to the General Sales terms available on Vitrolifes homepage (www.vitrolife.com)
and in cases of conflicting contents, the General Sales Terms prevails.
All IVF Media and Swemed IVF Instruments carry the European CE mark. With two
exceptions (CCM and Stem Cell Cutting Tool) all products are also cleared for sale in
the US.
The information in this manual was correct at the time of printing.
Latest updates can be found at www.vitrolife.com
Correspondence
Vitrolife welcomes any comments on the text in the Manual.
If you have questions or need further explanations, please contact us:
Europe, Middle East, Asia/Pacific
Tel:
+46-31-721 80 20 support line
Fax:
+46-31-721 80 99
E-mail: support.fertility@vitrolife.com
Address: Vitrolife Sweden AB

Box 9080

SE-400 92 Gteborg
Sweden
Internet
http://www.vitrolife.com
Americas
Tel:
866-VITRO US (866-848-7687)
Fax:
866-VITROFAX (866-848-7632)
E-mail: support.us.fertility@vitrolife.
com
Address: Vitrolife Inc.

3601 South Inca Street
Englewood

Colorado 80110
USA
Correspondence
65
66
67
Americas
Vitrolife Inc., 3601 South Inca Street
Englewood, Colorado 80110, USA
Tel 866-848-7687 (866-VITRO US)
Fax 866-848-7632 (866-VITROFAX)
Europe, Middle East, Asia/Pacific
Vitrolife Sweden AB, Box 9080
SE-400 92 Gteborg, Sweden
Tel +46-31-721 80 20, Fax +46-31-721 80 99
E-support Americas
support.us.fertility@vitrolife.com
E-support Europe, Middle East, Asia/Pacific
support.fertility@vitrolife.com
E-mail
fertility@vitrolife.com
Internet
http://www.vitrolife.com

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G-SERIES Manual

Recommended use of G-Series by Gardner

20022013 Vitrolife Sweden AB. All rights reserved.

Vitrolife G-Series Manual 6.0

Recommended use of G-MOPS 8

Vitrolife G-Series Manual 6.0

G-Series and related products

Media contents G-Series

Precautions and warnings

The Vitrolife Fertility Products

Swemed IVF instruments by Vitrolife

Vitrolife G-Series Manual 6.0

Vitrolife G-Series Manual 6.0

Aseptic working techniques means to work in the absence of microorganisms

Supplementing media with G-MM

Vitrolife G-Series Manual 6.0

Supplementation of G-MOPS, G-1, G-2, G-PGD.

Vitrolife G-Series Manual 6.0

G-FreezeKit Blast and G-ThawKit Blast require the following

Recommended use of G-MOPS /G-MOPS PLUS

Vitrolife G-Series Manual 6.0

G-MOPS for oocyte aspiration

G-MOPS for gamete and embryo handling

Vitrolife G-Series Manual 6.0

Pipette pre-warmed G-MOPS/G-MOPS PLUS into a culture dish placed in a

Vitrolife G-Series Manual 6.0

Hands on suggested procedure of pH measurement

a) Sintered disk window

IVF and Culture system

Vitrolife G-Series Manual 6.0

Vitrolife G-Series Manual 6.0

Swim-up method (migration)

Liquify for 20 minutes

Liquify for 20 minutes

Incubate in 37C, 6 % CO2 for 3060 minutes.

Vitrolife G-Series Manual 6.0

Remove top medium. Dilute with G-IVF / G-IVF PLUS. Centrifuge.

6 Discard the supernatant and re-suspend the pellet in 5.0 mL of equilibrated

8 Determine motility and concentration of spermatozoa in the washed sample.

equilibrate at +37C and 6 % CO2 for at least 2 hours.

Alternatively: Add equilibrated sperm suspension to equilibrated dishes with the

If oil overlay is used, droplets of at least 100 L volume are recommended.

DENS B,C,D GIII

c) for 10 minutes at 300600g.

C. Wash pellet with

7 Dilute the washed sample with equilibrated supplemented G-IVF/

Alternatively: Add equilibrated sperm suspension to equilibrated dishes with the

Vitrolife G-Series Manual 6.0

MICRO SETUP A,B

Vitrolife G-Series Manual 6.0

Vitrolife G-Series Manual 6.0

Pre-warmed G-MOPS is used as the rinsing fluid for the procedure.

Before use, ensure that the temperature of the media is +37C.

Vitrolife G-Series Manual 6.0

Oocyte culture and fertilisation

Vitrolife G-Series Manual 6.0

Alternatively: Add equilibrated sperm suspension to equilibrated dishes with the

Hands on fertilisation assessment

Vitrolife G-Series Manual 6.0

Scoring of human pronuclear embryos

Vitrolife G-Series Manual 6.0

Small volumes (droplets 0.05 mL 0.1 mL) under oil

A. Multi-well with OVOIL

B. Droplets under oil