Recommended use of G-Series⢠by Gardner Edition 7 ... - Vitrolife
Recommended use of G-Series⢠by Gardner Edition 7 ... - Vitrolife
Recommended use of G-Series⢠by Gardner Edition 7 ... - Vitrolife
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G-SERIES Manual<br />
<strong>Recommended</strong> <strong>use</strong> <strong>of</strong> G-Series <strong>by</strong> <strong>Gardner</strong><br />
<strong>Edition</strong> 7, January 2013
© 2002–2013 <strong>Vitrolife</strong> Sweden AB. All rights reserved.<br />
You may copy this for internal <strong>use</strong> only, not for publishing.<br />
The <strong>Vitrolife</strong> logotype is a trademark <strong>of</strong> <strong>Vitrolife</strong> Sweden AB, registered in Europe, the<br />
U.S. and other countries.<br />
<strong>Vitrolife</strong> Sweden AB<br />
Box 9080<br />
SE-400 92 Göteborg<br />
Sweden<br />
Tel: +46-31-721 80 00<br />
<strong>Vitrolife</strong> Inc.<br />
3601 South Inca Street<br />
Englewood<br />
Colorado 80110<br />
USA<br />
Tel: +1-866-VITRO US (866-848-7687)<br />
G5, G-Series, the G5 logo, GIII, GIII Series, the GIII logo, G-MM, HSAsolution,<br />
G-IVF, G-IVF PLUS, G-1, G-1 PLUS, G-2, G-2 PLUS,<br />
G-GAMETE, G-MOPS, G-MOPS PLUS, G-RINSE, G-FreezeKit Blast,<br />
G-ThawKit Blast, G-PGD, SpermGrad, HYASE, ICSI, FreezeKit Cleave,<br />
ThawKit Cleave and V-Tip are trademarks <strong>of</strong> <strong>Vitrolife</strong> Sweden AB.<br />
EmbryoGlue® is a registered trademark <strong>of</strong> <strong>Vitrolife</strong> Sweden AB.<br />
2<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
Contents<br />
Introduction 4<br />
General 5<br />
Opening your <strong>Vitrolife</strong> products 5<br />
CO 2 equilibration 6<br />
Supplementing media 6<br />
<strong>Recommended</strong> <strong>use</strong> <strong>of</strong> G-MOPS 8<br />
How to measure pH 10<br />
IVF and Culture System 12<br />
Sperm preparation 13<br />
Swim-up method (migration) 14<br />
Density gradient centrifugation method 16<br />
IVF-ET 19<br />
Follicle aspiration 19<br />
Oocyte identification 20<br />
Oocyte culture and fertilisation 21<br />
Insemination 22<br />
Fertilisation assessment 22<br />
Scoring <strong>of</strong> human pronuclear embryos 23<br />
Choosing the culture system 24<br />
Preparation <strong>of</strong> culture system 25<br />
Embryo culture 26<br />
Embryo assessment 28<br />
Scoring criteria for human blastocysts 29<br />
Scoring system 30<br />
Table for assessment <strong>of</strong> blastocysts 31<br />
Blastocyst schedule 32<br />
Embryo transfer 33<br />
Replacement <strong>of</strong> embryos 33<br />
Loading the catheter 35<br />
Micromanipulation 37<br />
Denudation <strong>of</strong> oocytes for ICSI 38<br />
ICSI procedure 40<br />
Difficult ICSI cases 42<br />
Testicular biopsy 43<br />
Embryo biopsy 43<br />
Embryo Cryopreservation 45<br />
Equipment list 45<br />
Cryopreservation <strong>of</strong> cleavage<br />
stage embryos using Freezekit Cleave<br />
and Thawkit Cleave 47<br />
Cryopreservation <strong>of</strong> cleavage<br />
stage embryos using Freezekit 1<br />
and Thawkit 1 49<br />
Cryopreservation <strong>of</strong> blastocyst stage<br />
embryos 52<br />
Quality Control Program 56<br />
G-Series and related products<br />
from <strong>Vitrolife</strong> 59<br />
Media contents G-Series 61<br />
Instruments <strong>by</strong> <strong>Vitrolife</strong> 62<br />
Precautions and warnings 64<br />
Correspondence 65<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 Contents 3
Introduction<br />
Improving success rates<br />
<strong>Vitrolife</strong> is dedicated to improve the success rates <strong>of</strong> Human Assisted Reproduction. Long<br />
term research in reproductive physiology and studies <strong>of</strong> embryo development has resulted<br />
in the most advanced IVF media products available. This manual describes the <strong>use</strong> <strong>of</strong><br />
<strong>Vitrolife</strong>’s G-Series <strong>by</strong> Dr. David K. <strong>Gardner</strong>.<br />
We are well aware that there are many ways <strong>of</strong> practicing assisted reproductive technology.<br />
The methods we describe below have resulted in high success rates with our products.<br />
The <strong>Vitrolife</strong> Fertility Products<br />
IVF media<br />
<strong>Vitrolife</strong> <strong>of</strong>fers IVF media products <strong>of</strong> the highest efficacy and safety. Every LOT undergoes<br />
rigorous Quality Control testing including mo<strong>use</strong> embryo assay (MEA) before release to<br />
customers. With every released LOT, a Certificate <strong>of</strong> Analysis is issued.<br />
Quality Control together with quality assured operations (ISO 13485:2003 and 21 CFR<br />
Part 820:QSR) guarantees LOT-to-LOT consistency. All raw materials <strong>use</strong>d for the manufacturing<br />
<strong>of</strong> <strong>Vitrolife</strong> Fertility Products are selected as the best available on the world market.<br />
All raw materials are tested and evaluated individually <strong>by</strong> stringent quality control and MEA<br />
procedures before <strong>use</strong> in the manufacturing <strong>of</strong> our media.<br />
Swemed IVF instruments <strong>by</strong> <strong>Vitrolife</strong><br />
<strong>Vitrolife</strong> also provides a number <strong>of</strong> instruments for the IVF treatment, such as aspiration<br />
needles, denudation pipettes, pipettes for ICSI and embryo biopsy, and transfer catheters.<br />
Like the media, the instruments undergo a strict quality control and are manufactured in<br />
accordance with the same Quality Standards as the media.<br />
We are certain you will agree that the “creation” <strong>of</strong> new human life deserves the best possible<br />
environment. We are committed to bringing you the highest quality products.<br />
4<br />
Introduction<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
General<br />
Opening your <strong>Vitrolife</strong> products<br />
When you receive your delivery <strong>of</strong> <strong>Vitrolife</strong> products you may notice that they are packaged<br />
in a special way. There are specific reasons for this:<br />
• All <strong>Vitrolife</strong> products are tamper-evident sealed. The packaging ensures that it is<br />
impossible to enter the bottle without visible evidence.<br />
• All materials <strong>use</strong>d in the packaging are non-toxic so that nothing may interfere with the<br />
final product. PETG bottles, screw caps, specially designed labels and pharmaceutical<br />
sealing are all part <strong>of</strong> this tamper evident protocol.<br />
G-Series PLUS media, except G-IVF PLUS, are supplemented with 5 mg HSA/mL.<br />
G-IVF PLUS, is supplemented with 10 mg HSA/mL. All other G5 media are protein-free<br />
(unless otherwise labeled) and need to be supplemented with either G-MM (recombinant<br />
human albumin) or HSA-solution (human serum albumin) according to bottle labels. See<br />
Table on page 7.<br />
Essentials<br />
• Wash and disinfect your hands before handling any product.<br />
• Take necessary precautions when handling any biological fluid such as blood, follicular<br />
fluid and semen.<br />
Hands on<br />
1 Open all products in a clean laminar air flow (LAF) cabinet.<br />
2 Before bringing bottles into the LAF cabinet, assure the bottles are clean on the<br />
outside. It is recommended that the bottles are wiped with a lint free cloth and ethanol.<br />
3 Identify the product and check the expiry date. Break the tamper-evident seal and<br />
discard.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 General 5
4 Remove the cap and place the cap face down in a sterile petri dish.<br />
5 Remove the desired volume with a sterile non-toxic pipette. Replace bottle cap and<br />
screw on firmly. Do not touch the inner sides <strong>of</strong> the cap.<br />
6 Keep the media cold as much as possible. Prepare dishes immediately after the bottles<br />
have been removed from the refrigerator. Media bottles should not be left at ambient<br />
temperature for a longer period <strong>of</strong> time than it takes to prepare dishes or tubes.<br />
Aseptic working techniques means to work in the absence <strong>of</strong> microorganisms<br />
capable <strong>of</strong> causing infection or contamination.<br />
CAUTION<br />
Never pour the contents out <strong>of</strong> the bottle, as the lip <strong>of</strong> the bottle may not be<br />
sterile once opened.<br />
CO 2 equilibration<br />
Optimal pH for culture <strong>of</strong> human embryos is 7.2- 7.4.<br />
To obtain correct pH, all G-Series media (with the exception <strong>of</strong> G-MOPS /G-MOPS <br />
PLUS, G-PGD , G-Freezekit Blast and G-Thawkit Blast ), should be equilibrated at 6%<br />
CO 2 . This is a general recommendation that is valid for laboratories located at or near sea<br />
level. For laboratories located at higher altitudes, the CO 2 percentage should be increased<br />
<strong>by</strong> approximately 0.6% CO 2 per 1000 meters.<br />
Correct pH must always be verified with pH measurements. See page 10.<br />
Supplementing media with G-MM <br />
or HSA-solution <br />
In this manual the word “supplemented” is frequently <strong>use</strong>d. Supplemented<br />
means the addition <strong>of</strong> G-MM or HSA-solution as described in this section, or<br />
G-Series PLUS products.<br />
G-Series PLUS media, G-RINSE, G-GAMETE and EmbryoGlue® do not<br />
require albumin supplementation.<br />
6<br />
General<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
All other G-Series culture media, not designated PLUS, come protein-free and<br />
need to be supplemented either with G-MM or HSA-solution at the appropriate<br />
concentration as listed in the table below.<br />
When selecting protein supplementation it should be noted that Human Serum<br />
Albumin (HSA) is a blood derived substance and may contain human pathogenic<br />
agents including those not yet known or identified. Thus the risk <strong>of</strong> transmission<br />
<strong>of</strong> such infectious agents cannot be completely eliminated when using HSA.<br />
G-MM containing recombinant human albumin in place <strong>of</strong> HSA may contain<br />
an extremely small amount <strong>of</strong> yeast antigens (less than 0.15ppm) from the<br />
manufacturing process. Since no animal or human derived raw materials are <strong>use</strong>d<br />
in the manufacturing process <strong>of</strong> recombinant human albumin, all human or animal<br />
derived infectious agents are proscribed <strong>by</strong> virtue <strong>of</strong> the biosynthetic character <strong>of</strong><br />
the product.<br />
G-Series media with the exception <strong>of</strong> G-IVF shall have 5 % <strong>of</strong> either G-MM or HSAsolution<br />
added (0.5 mL added to 9.5 mL). The final concentration <strong>of</strong> G-MM will be 2.5<br />
mg/mL and the final concentration <strong>of</strong> HSA will be 5 mg/mL.<br />
G-IVF shall have 10 % <strong>of</strong> either G-MM or HSA-solution added (1.0 mL added to 9.0<br />
mL). The final concentration <strong>of</strong> G-MM will be 5 mg/mL and the final concentration <strong>of</strong> HSA<br />
will be 10 mg/mL.<br />
Media to be supplemented should be aliquoted into sterile non-toxic tissue culture grade<br />
tubes or flasks. All containers should be pre-rinsed using G-RINSE in order to ensure<br />
there is no particulate matter or toxic substances residing.<br />
Supplementation <strong>of</strong> G-MOPS , G-1 , G-2 , G-PGD .<br />
Medium [mL] G-MM or HSA-solution [mL] Final volume [mL]<br />
9.5 0.5 10.0<br />
19.0 1.0 20.0<br />
28.5 1.5 30.0<br />
38.0 2.0 40.0<br />
47.5 2.5 50.0<br />
57.0 3.0 60.0<br />
66.5 3.5 70.0<br />
76.0 4.0 80.0<br />
85.5 4.5 90.0<br />
95.0 5.0 100.0<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
General<br />
7
Supplementation <strong>of</strong> G-IVF<br />
Medium [mL] G-MM or HSA-solution [mL] Final volume [mL]<br />
9.0 1.0 10.0<br />
18.0 2.0 20.0<br />
27.0 3.0 30.0<br />
36.0 4.0 40.0<br />
45.0 5.0 50.0<br />
54.0 6.0 60.0<br />
63.0 7.5 70.0<br />
72.0 8.0 80.0<br />
81.0 9.0 90.0<br />
90.0 10.0 100.0<br />
G-FreezeKit Blast and G-ThawKit Blast require the following<br />
supplementation (please read pages 50 and 52):<br />
9.0 mL Freeze/Thaw solution + 1.0 mL G-MM or 1.0 mL HSA-solution <br />
The cryopreservation solutions can be supplemented directly into the dishes. Simply place<br />
900 µL <strong>of</strong> each cryopreservation solution into separate wells + 100 µL <strong>of</strong> G-MM or 100 µL<br />
HSA-solution .<br />
It is recommended only to supplement the volume <strong>of</strong> medium expected to be<br />
<strong>use</strong>d in one day.<br />
<strong>Recommended</strong> <strong>use</strong> <strong>of</strong> G-MOPS /G-MOPS PLUS<br />
G-MOPS /G-MOPS PLUS are intended for handling <strong>of</strong> gametes and embryos outside <strong>of</strong><br />
the CO 2 incubator.<br />
Essentials<br />
In order not to subject oocytes and embryos to unnecessary stress when working outside <strong>of</strong><br />
the CO 2 incubator, it is very important to work as quickly as possible. As the lid <strong>of</strong> the dishes<br />
must be removed when moving oocytes and embryos between dishes and when performing<br />
ICSI, pH as well as temperature and osmolality may change after some time. This is valid for<br />
all media including G-MOPS /G-MOPS PLUS.<br />
8<br />
General<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
Importance <strong>of</strong> pH<br />
G-MOPS /G-MOPS PLUS are MOPS buffered media and must be <strong>use</strong>d at +37°C in<br />
an air atmosphere. Do not put this medium in a CO 2 environment as the pH will go down<br />
below the specification range. Furthermore, do not <strong>use</strong> paraffin oil equilibrated in a CO 2<br />
environment when covering G-MOPS /G-MOPS PLUS.<br />
Importance <strong>of</strong> Temperature<br />
Oocytes and embryos must be kept at +37°C at all times.<br />
To ensure the temperature <strong>of</strong> G-MOPS /G-MOPS PLUS inside <strong>of</strong> a tube/dish is +37°C,<br />
it is recommended to validate the procedure using a certified thermometer that is placed<br />
inside <strong>of</strong> a tube/dish containing a sample amount <strong>of</strong> G-MOPS /G-MOPS PLUS, in the<br />
warming block (tube) or on a heated stage (dish). The goal is to ensure the G-MOPS /G-<br />
MOPS PLUS is +37°C before <strong>use</strong>. Individual pieces <strong>of</strong> warming equipment should be<br />
adjusted to attain this goal.<br />
Importance <strong>of</strong> Osmolality<br />
To avoid changes in osmolality, tubes must be filled as much as possible and tightly capped.<br />
Dishes should be covered with oil or kept with the lid on when not in <strong>use</strong>.<br />
G-MOPS for oocyte aspiration<br />
G-MOPS for oocyte aspiration should not to be supplemented. In order to ensure the<br />
G-MOPS is appropriately warmed to +37°C before <strong>use</strong>, pre-warm the medium <strong>by</strong> placing<br />
a tightly sealed bottle <strong>of</strong> G-MOPS into a warming incubator without CO 2 . The bottle must<br />
be tightly capped to avoid changes in osmolality. It can take up to 4 hours for a bottle <strong>of</strong><br />
G-MOPS to reach +37°C. Each time the bottle is removed from the warming incubator<br />
and <strong>use</strong>d, ensure the cap is tightly replaced. Discard any G-MOPS that is warmed and<br />
not <strong>use</strong>d within the recommended time frame, i.e. the same day if the medium is stored in a<br />
tightly capped tube until <strong>use</strong> or after 60 min if the medium volume is minimum 1 mL in a dish<br />
and not covered with oil. A dish covered with oil can be <strong>use</strong>d for up to 2 hours provided that<br />
the appropriate temperature <strong>of</strong> the heating stage is maintained.<br />
G-MOPS for gamete and embryo handling<br />
G-MOPS for gamete and embryo handling must to be supplemented. In a laminar flow<br />
hood pipette G-MOPS /G-MOPS PLUS into non toxic sterile test tubes. If G-MOPS is<br />
<strong>use</strong>d supplement with the appropriate amount <strong>of</strong> G-MM or HSA-solution . The tube must<br />
be filled and tightly capped to avoid changes in osmolality. Place the tube in a warming<br />
incubator without CO 2 or in an adequately calibrated warming block. Pipette pre-warmed<br />
G-MOPS /G-MOPS PLUS into a pre-warmed culture dish. To avoid changes in osmolality<br />
<strong>use</strong> the medium as soon as possible after preparation, i.e. 60 minutes if the dish is not<br />
covered with oil (minimum 1 mL <strong>of</strong> medium) or 2 hours if the dish is covered with oil.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
General<br />
9
Pipette pre-warmed G-MOPS/G-MOPS PLUS into a culture dish placed in a<br />
warming incubator without CO 2 or on a heated stage, with the lid on. To avoid<br />
changes in osmolality, cover with oil or <strong>use</strong> the dish within 60 minutes after<br />
preparation. This statement is valid for a volume <strong>of</strong> 1 mL or higher. To ensure<br />
that the temperature inside <strong>of</strong> the culture dish is +37°C it is recommended<br />
to validate the procedure using a certified thermometer that is placed inside<br />
<strong>of</strong> a culture dish with G-MOPS/G-MOPS PLUS on a heated stage. The<br />
temperature setting <strong>of</strong> the heated stage needs to be adjusted to achieve the<br />
correct +37°C <strong>of</strong> the medium in the culture dish.<br />
IMPORTANT<br />
Ensure that no G-MOPS/G-MOPS PLUS is transferred to the culture dishes<br />
<strong>by</strong> introducing washing steps between the G-MOPS dish and the culture<br />
dish. The washing procedure should include at least two steps <strong>of</strong> 1 mL culture<br />
medium per step. The wash dishes should be changed after 5 oocytes or<br />
embryos.<br />
How to measure pH<br />
Introduction<br />
Measurement <strong>of</strong> pH with a glass membrane electrode and a modern instrument is a<br />
straightforward process. There are however several pitfalls to be avoided when the highest<br />
possible accuracy needs to be achieved. This is especially the case if pH must be measured<br />
in solutions with dissolved gas or with high protein content.<br />
Essentials<br />
• Use a semi-micro or a micro electrode with a built in reference electrode and<br />
temperature measurement.<br />
• Set the instrument for automatic temperature compensation.<br />
• Use fresh buffers with at least one having a pH close to that <strong>of</strong> the tested solution.<br />
Change the buffer between every test.<br />
• Confirm the proper function <strong>of</strong> the electrode daily <strong>by</strong> testing slope and <strong>of</strong>fset.<br />
• Change the electrode as soon as it starts to deteriorate. A pH-electrode rarely lasts for<br />
longer than 12 months and may <strong>of</strong>ten have to be replaced every second or third month.<br />
• Store the electrode in a neutral buffer or in a special storage solution.<br />
• Calibrate the instrument before each sequence <strong>of</strong> testing.<br />
10<br />
General<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
Hands on – suggested procedure <strong>of</strong> pH measurement<br />
(The exact procedure may vary depending on instrumentation and the nature <strong>of</strong> the sample)<br />
1 Take the electrode from the storage solution, rinse it and place it in pH 7.00 buffer.<br />
Remove the plug or tube that blocks the air inlet to the electrode.<br />
2 Let the electrode stand in the pH 7.00 buffer for between one and two hours. This time<br />
may be reduced if the electrode is stored in pH 7.0 buffer over night.<br />
3 Calibrate the electrode using two fresh buffers, one close to pH 7.00 and one at e.g. pH<br />
10.0.<br />
4 Verify that the slope and the <strong>of</strong>fset <strong>of</strong> the electrode are within the specified limits <strong>of</strong><br />
the electrode manufacturer. We recommend that the slope should be between 95 and<br />
102 % <strong>of</strong> the theoretical value, and that the <strong>of</strong>fset at pH 7.00 should not differ more than<br />
± 4 mV from the value acquired during the previous calibration.<br />
5 Verify that the calibration was performed properly <strong>by</strong> testing a buffer with a pH <strong>of</strong><br />
approximately 8.00. Compare the measured and the specified pH at the actual test<br />
temperature. The measured pH may not differ from the specified pH <strong>by</strong> more than<br />
± 0.05 pH units.<br />
6 Place the electrode in distilled water at approximately the same temperature that the<br />
sample will have when it is tested.<br />
7 Equilibrate the sample at the temperature and gas composition that is required.<br />
Equilibration with CO 2 may take up to 16 hours!<br />
8 Take the electrode from the distilled water; wipe the tip <strong>of</strong> the electrode briefly with<br />
a s<strong>of</strong>t clean tissue to remove the water. Place the electrode immediately in the<br />
equilibrated media. The glass bulb and the contact point <strong>of</strong> the reference electrode<br />
PH measure<br />
must be covered with the equilibrated media, see a) and b) below.<br />
a) b)<br />
a) Sintered disk window b) Sleeve joint<br />
SWIMUP C<br />
a)<br />
b)<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
General<br />
11
9 Take a reading from the pH instrument as soon as the pH is stable. If the test media<br />
has been equilibrated with CO 2 the entire test procedure must be completed within 30<br />
seconds from the removal <strong>of</strong> the media from the incubator. If the test takes longer time<br />
than this, CO 2 will dissipate from the sample into the atmosphere and pH in the medium<br />
will rise.<br />
10 As an extra precaution a control medium with known pH at the conditions at hand may<br />
be tested in parallel with the unknown sample. This enables a confirmation to be made<br />
that the entire test procedure was performed correctly.<br />
11 Register all data from the test, including the calibration data, in appropriate records or<br />
log-books.<br />
IVF and Culture system<br />
We are aware that there are many options to choose from when considering the culture<br />
system for IVF. This manual provides a suggested methodology that has been proven successful<br />
for our products.<br />
Essentials<br />
• Choose your culture systems for their efficacy, simplicity and reproducibility.<br />
• Organise yourself and prepare in advance.<br />
• Use only non-toxic sterile disposables that are subject to quality control for embryo<br />
culture and keep a log <strong>of</strong> all items and procedures performed.<br />
• Adhere to your protocols to ensure consistency.<br />
• Ensure that all <strong>of</strong> your equipment is correctly maintained, and regularly checked and<br />
calibrated.<br />
• All procedures should be performed in a clean, dedicated work environment<br />
(LAF cabinet).<br />
• Use aseptic working techniques.<br />
• Qualify all operators for handling media and keep training protocols consistent.<br />
• Always wear non-toxic gloves when handling biological fluids.<br />
• Check the identity <strong>of</strong> the patient and label all materials before proceeding.<br />
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<strong>Vitrolife</strong> G-Series Manual 6.0
Sperm<br />
Preparation<br />
Prior to being <strong>use</strong>d for IUI, IVF and ICSI, motile sperm cells are separated from the seminal<br />
plasma, dead sperm cells and other cells. This can be done <strong>by</strong> different procedures.<br />
The decision <strong>of</strong> which semen preparation method to <strong>use</strong> is based on the patient history, previous<br />
semen analyses, as well as an examination <strong>of</strong> the present sample. Another consideration is whether<br />
fertilisation will be achieved <strong>by</strong> IVF or ICSI. In IVF you will need more sperm for insemination. The<br />
preparation methods select sperm based on their motility, ideally selecting only live sperm, or on<br />
their density, ideally selecting only mature sperm. If the sperm count and motility are adequate,<br />
migration (swim-up) is suitable. If semen quality is poor and includes large numbers <strong>of</strong> other cells,<br />
density gradient centrifugation is preferred. Recovery <strong>of</strong> sperm is more effective using the gradient<br />
centrifugation method rather than using the swim-up procedure, with respect to total yield. However,<br />
in some instances percent motility can be higher in sperm prepared <strong>by</strong> swim-up.<br />
Essentials<br />
• The sperm preparation should be performed in a clean aseptic work area. Non-toxic,<br />
non-powdered gloves and protective eye glasses should be worn while handling semen<br />
samples.<br />
• All samples should be collected in appropriate sterile, non-toxic vessels. It is<br />
recommended that the semen sample be collected not more than one hour before<br />
preparation. The semen sample should be protected from cold and heat.<br />
• All laboratory procedures, including a thorough identification protocol <strong>of</strong> the patient<br />
should be followed.<br />
• Sterile, non-toxic tubes, needles and pipettes should be washed with G-RINSE before<br />
<strong>use</strong> for the preparation <strong>of</strong> sperm.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
Sperm Preparation<br />
13
Swim-up method (migration)<br />
Hands on<br />
This method is to be <strong>use</strong>d for the semen samples with a good sperm count and motility.<br />
1 Allow approximately 20 minutes for liquefaction <strong>of</strong> semen. If the sample does not liquefy,<br />
you may need to pass it through a 23 gauge needle or a non-toxic sterile narrow Pasteur<br />
pipette.<br />
Liquify for 20 minutes<br />
Liquify for 20 minutes<br />
2 Make a microscopic assessment <strong>of</strong> the sperm sample to confirm the optimal method for<br />
processing the sperm.<br />
PH measure<br />
a) b)<br />
3 Mark a test tube with patient ID. More than one tube can be set up if there are concerns<br />
about semen quality.<br />
4 Pipette 1.0 mL <strong>of</strong> semen into a rinsed tube. Make up 2-4 tubes depending on semen<br />
volume. Carefully overlay 2.0 mL <strong>of</strong> pre-equilibrated supplemented G-IVF /G-IVF PLUS.<br />
Place the swim-up tube in an angled position in the incubator at +37°C and 6 % CO 2 for<br />
SWIMUP C<br />
30–60 minutes.<br />
a)<br />
b)<br />
a)<br />
b)<br />
Incubate in 37°C, 6 % CO 2 for 30–60 minutes.<br />
a) G-IVF / G-IVF PLUS b) Semen<br />
SWIMUP D/E<br />
14<br />
Sperm Preparation<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
)<br />
5 Aspirate the top medium without touching the underlaying semen and transfer to a clean<br />
tube. SWIMUP Add 5.0 D/E mL <strong>of</strong> equilibrated supplemented G-IVF /G-IVF PLUS, mix and centrifuge<br />
for 10 minutes at 300–600 g.<br />
Remove top medium. Dilute with G-IVF / G-IVF PLUS. Centrifuge.<br />
6 Discard the supernatant and re-suspend the pellet in 5.0 mL <strong>of</strong> equilibrated<br />
supplemented G-IVF /G-IVF PLUS and repeat the centrifugation at 300–600 g for 10<br />
min. DENS A GIII<br />
A<br />
Remove<br />
supernatant.<br />
Repeat a) wash.<br />
b)<br />
c)<br />
Discard supernatant. Repeat wash.<br />
7 Discard the supernatants and combine all pellets. Re-suspend the pellets in 0.5–1.0 mL<br />
<strong>of</strong> DENS equilibrated B,C,D supplemented GIII G-IVF /G-IVF PLUS, depending on sample quality.<br />
B<br />
Resuspend in<br />
C D<br />
small volume<br />
G-FERT.<br />
Resuspend a) in small volume G-IVF / G-IVF PLUS.<br />
8 Determine motility and concentration <strong>of</strong> spermatozoa in the washed sample.<br />
9 Dilute the washed sample with equilibrated supplemented G-IVF /G-IVF PLUS to a<br />
final PREPCULT concentration A,B<strong>of</strong> 75 000–200 000 motile sperm/mL.<br />
10 Prepare rinsed insemination dishes with 0.5-1.0 mL <strong>of</strong> sperm suspension and<br />
A<br />
a)<br />
B<br />
a)<br />
equilibrate at +37°C and 6 % CO 2 for at least 2 hours.<br />
Alternatively: Add equilibrated sperm suspension to equilibrated dishes with the<br />
oocytes already present.<br />
b)<br />
b)<br />
If oil overlay is <strong>use</strong>d, droplets <strong>of</strong> at least 100 µL volume are recommended.<br />
MICRO A,B<br />
A a)<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
f)<br />
b)<br />
a) b)<br />
Sperm Preparation<br />
15
Density<br />
PH measure<br />
gradient centrifugation method<br />
This method can be <strong>use</strong>d to wash all samples <strong>of</strong> sperm regardless <strong>of</strong> quality.<br />
SpermGrad is a solution <strong>of</strong> silane coated, colloidal silica particles in an isotonic balanced salt<br />
solution. By using a) different dilutions <strong>of</strong> SpermGrad, solutions b) <strong>of</strong> different densities are obtained.<br />
Layering these solutions <strong>of</strong> different densities carefully in a centrifuge tube creates a density gradient.<br />
Cells and other particles with different buoyant densities will sediment until they reach a solution<br />
with higher density. Centrifugation accelerates this sedimentation. Commonly, a two-step gradient<br />
<strong>of</strong> 90 % and 45 % SpermGrad is <strong>use</strong>d. Since mature sperm with tightly packed DNA have a higher<br />
density than 90 % SpermGrad, they sediment through this layer and are found at the bottom <strong>of</strong> the<br />
tube, whereas other cells, including immature and dead sperm, stop sedimenting at the 90 % or 45<br />
% SWIMUP interface. You Ccan <strong>use</strong> either SpermGrad RTU solutions already diluted to 90% (Upper Layer)<br />
and 45% (Lower Layer) or SpermGrad 100% stock solution, should you prefer to make your own<br />
dilutions. The stock solution must be diluted in G-IVF/G-IVF PLUS into appropriate concentrations.<br />
G-IVF should be supplemented with either G-MM or HSA-solution. G-IVF/G-IVF<br />
PLUS must be equilibrated at +37 °C and 6% CO 2 before <strong>use</strong>.<br />
Hands on<br />
b)<br />
1 If you <strong>use</strong> SpermGrad RTU, continue to paragraph 2. If you <strong>use</strong> SpermGrad stock<br />
solution:<br />
Mix SpermGrad with supplemented G-IVF /G-IVF PLUS in separate tubes to obtain<br />
90 SWIMUP % and 45 D/E % stock solutions. For 90 % stock solution, mix 9.0 mL SpermGrad with<br />
1.0 mL supplemented G-IVF . For 45 % stock solution, mix 4.5 mL SpermGrad with<br />
5.5 mL supplemented G-IVF . Mix the solutions thoroughly and store in sterile non-toxic<br />
tubes or sterile tissue culture flasks. Label and refrigerate until <strong>use</strong>. Always <strong>use</strong> a sterile<br />
non-toxic pipette to aliquot amounts needed for individual sperm preparations. Stock<br />
solutions should be labeled with the date and kept for recommended time frames (see<br />
expiry on bottles). Before <strong>use</strong>, allow the solutions to reach ambient temperature.<br />
2 The density gradient should be layered in 2 to 4 sterile and rinsed conical non-toxic<br />
centrifuge tubes (depending on the volume <strong>of</strong> the semen sample) marked with patient<br />
ID. Pipette 1.5 mL <strong>of</strong> 90 % solution into the tube first and then slowly pipette 1.5 mL <strong>of</strong><br />
45 % solution on top <strong>of</strong> it. Finally, 1.0 mL <strong>of</strong> the semen is gently layered on the top (A).<br />
Make up 2-4 gradient tubes. Up to 2 mL <strong>of</strong> semen can be layered on top <strong>of</strong> the gradient.<br />
If the DENS semen A GIII sample is <strong>of</strong> normal quality reduce the semen volume. Adding too much<br />
semen will result in poor separation.<br />
A<br />
a)<br />
a)<br />
b)<br />
c)<br />
A. Layer gradient solutions and semen in conical centrifuge tube. Spin 300–600g / 20 minutes.<br />
a) Semen b) 45 % c) 90 %<br />
DENS B,C,D GIII<br />
16<br />
Sperm Preparation B C<br />
<strong>Vitrolife</strong> D G-Series Manual 6.0
DENS A GIII<br />
3 The tubes are then centrifuged for 10–20 minutes at 300–600g.<br />
A<br />
4 Remove the two top layers and take care not to leave any residues on the tube wall (B).<br />
Transfer the sperm pellets with as little <strong>of</strong> the 90 % solution as possible to a sterile<br />
a)<br />
conical rinsed tube with 5 mL <strong>of</strong> equilibrated supplemented G-IVF /G-IVF PLUS.<br />
b)<br />
5 Centrifuge c) for 10 minutes at 300–600g.<br />
6 Aspirate and discard the supernatants and repeat the wash (C). After the second wash,<br />
combine pellets and re-suspended in 1 mL <strong>of</strong> equilibrated supplemented<br />
G-IVF DENS /G-IVF B,C,D PLUS GIII (D). The washed sample is then assessed for motility and<br />
concentration.<br />
B C D<br />
a)<br />
B. Remove top layers C. Wash pellet with D. Resuspend pellet<br />
and transfer pellet G-IVF/G-IVF PLUS. in small volume<br />
to clean tube. REPEAT. <strong>of</strong> G-IVF/G-IVF PLUS.<br />
a) Pellet<br />
PREPCULT A,B<br />
7 Dilute the washed sample with equilibrated supplemented G-IVF /<br />
A<br />
G-IVF a)<br />
B<br />
a)<br />
PLUS to a final concentration <strong>of</strong> 75 000–200 000 motile sperm/mL.<br />
8 Prepare rinsed insemination dishes with 0.5–1.0 mL <strong>of</strong> sperm suspension and<br />
equilibrate at +37 ° C and 6 % CO 2 for at least 2 hours.<br />
b)<br />
b)<br />
Alternatively: Add equilibrated sperm suspension to equilibrated dishes with the<br />
oocytes already present.<br />
It MICRO is recommended A,B to inseminate in a volume <strong>of</strong> 0.5-1.0 mL without oil overlay. If oil<br />
overlay is <strong>use</strong>d, droplets <strong>of</strong> at least 100 μL volume are recommended.<br />
A<br />
f)<br />
a)<br />
b)<br />
a) b)<br />
c) d)<br />
e)<br />
c)<br />
d)<br />
B<br />
a)<br />
b)<br />
c)<br />
d)<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 Sperm Preparation 17<br />
MICRO SETUP A,B
18<br />
Sperm Preparation<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
IVF-ET<br />
<strong>Vitrolife</strong> has developed culture media for the individual stages <strong>of</strong> embryo development.<br />
G-IVF /G-IVF PLUS should be <strong>use</strong>d for in vitro fertilisation. For cleavage and culture <strong>of</strong> early embryos<br />
G-1 /G-1 PLUS should be <strong>use</strong>d. For culture from day 3 up to the blastocyst stage, G-2 /G-2 <br />
PLUS should be <strong>use</strong>d. After ICSI, embryos should be placed directly into G-1 /G-1 PLUS.<br />
Irrespective <strong>of</strong> stage, embryos should be transferred to the uterus using EmbryoGlue® or supplemented<br />
G2 or G-2 PLUS.<br />
Follicle aspiration<br />
The purpose <strong>of</strong> the oocyte collection procedure is to collect as many oocytes as quickly as<br />
possible and to minimise the exposure <strong>of</strong> those oocytes to non-physiological conditions.<br />
Important parameters are temperature, osmolality and pH. Any deviations from physiologic<br />
conditions may have deleterious effects on the ability <strong>of</strong> the oocyte to fertilise normally and<br />
achieve normal preimplantation development.<br />
Essentials<br />
• Day 0 is defined as the day <strong>of</strong> oocyte collection.<br />
• Identify the patient and check ID label on culture dishes and pipettes before proceeding.<br />
• Ensure that all surfaces are warmed and that all materials that may come in contact with<br />
the oocytes are sterile, non-toxic and <strong>of</strong> tissue culture quality, preferably mo<strong>use</strong> embryo<br />
tested.<br />
• Oocyte aspiration is performed with the <strong>use</strong> <strong>of</strong> an ultrasonically guided needle with a<br />
double or single lumen. For recommended aspiration needles, see Swemed Instruments<br />
<strong>by</strong> <strong>Vitrolife</strong> page 62, Follicle Aspiration needles, V-Tip . Negative pressure is typically<br />
applied to the needle lumen <strong>by</strong> a regulated aspiration pump.<br />
• High or uncontrolled negative pressures may damage oocytes.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 IVF-ET 19
• Pre-warmed G-MOPS is <strong>use</strong>d as the rinsing fluid for the procedure.<br />
• G-MOPS is a modified G-1 medium, buffered with MOPS and sodium bicarbonate to<br />
maintain pH at room atmosphere.<br />
• For collecting aspirates and follicle flushing pre-warmed G-MOPS can be <strong>use</strong>d<br />
protein-free due to the high protein content <strong>of</strong> follicular fluid.<br />
• For washing <strong>of</strong> oocytes prior to incubation in supplemented G-IVF /G-IVF PLUS, prewarmed<br />
supplemented G-MOPS /G-MOPS PLUS should be <strong>use</strong>d.<br />
• G-GAMETE equilibrated at +37°C and 6 % CO 2 can be <strong>use</strong>d as an alternative to<br />
supplemented G-MOPS /G-MOPS PLUS.<br />
• It is advised to prepare one wash dish for a maximum <strong>of</strong> five follicles.<br />
Hands on<br />
1 Pre-equilibrate G-RINSE at +37°C in an atmosphere <strong>of</strong> 6 % CO 2 .<br />
2 Warming <strong>of</strong> G-MOPS PLUS for oocyte wash: pipette the medium in rinsed tubes.<br />
Tightly cap the tubes and place them in a warming incubator without CO 2 at +37°C.<br />
3 Warming <strong>of</strong> un-supplemented G-MOPS for follicle flushing: pipette the medium in<br />
rinsed tubes. Tightly cap the tubes and place them in a warming incubator without CO 2<br />
at +37°C.<br />
Before <strong>use</strong>, ensure that the temperature <strong>of</strong> the media is +37°C.<br />
4 Rinse the needle lumen and tubing using G-RINSE and discard the rinsed fluid.<br />
5 The follicles may be aspirated individually or collectively. The aspirated follicular fluid<br />
must be collected in a rinsed sterile non-toxic tissue culture tube. G-MOPS can be<br />
supplemented with quality tested, pharmaceutical grade Heparin (2.5–10.0 units/mL)<br />
to reduce clotting <strong>of</strong> the follicular aspirates containing blood.<br />
6 The follicle aspirates should be collected in sterile non-toxic tissue culture disposable<br />
tubes and should be examined <strong>by</strong> the laboratory immediately. If they cannot be<br />
examined directly, the tubes should be tightly sealed and kept at +37°C.<br />
Oocyte identification<br />
Essentials<br />
• wear non-toxic gloves<br />
• identify the patient before proceeding<br />
• work rapidly to avoid cooling<br />
20<br />
IVF-ET<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
Hands on<br />
1 Follicle aspirates should be kept at +37°C. The <strong>use</strong> <strong>of</strong> a test tube heating block next<br />
to the microscope can facilitate this. Follicle aspirates are microscopically examined in<br />
sterile tissue culture Petri dishes.<br />
2 Should it be necessary to flush a follicle, G-MOPS pre-warmed to +37°C should be<br />
<strong>use</strong>d.<br />
3 Transfer the follicular aspirates to an empty dish. Identify the oocytes, pre-rinse a sterile<br />
pipette with supplemented G-MOPS /G-MOPS PLUS, and immediately remove the<br />
oocytes from the follicular fluid and possible blood contamination.<br />
4 Rinse the oocytes first in pre-warmed supplemented G-MOPS /G-MOPS PLUS,<br />
and then in equilibrated supplemented G-IVF /G-IVF PLUS. The washing procedure<br />
should include at least two steps with 1.0 mL <strong>of</strong> G-IVF /G-IVF PLUS in each step.<br />
Transfer the oocytes to the dishes with equilibrated supplemented G-IVF /G-IVF PLUS<br />
and return the dishes to the incubator immediately.<br />
Oocyte culture and fertilisation<br />
Essentials<br />
• Crucial considerations for gamete handling and fertilisation are temperature, pH and<br />
osmolality.<br />
• Temperature loss occurs rapidly and is related to the amount <strong>of</strong> time a culture vessel<br />
stays out <strong>of</strong> the incubator and whether the surface is heated, e.g. a heated microscope<br />
stage.<br />
• Changes in osmolality depend on volume, temperature and presence or absence <strong>of</strong> oil<br />
overlay. Changes in osmolality will occur slowly and is <strong>of</strong>ten an undetected parameter.<br />
The culture system should be correctly humidified and preferably under oil.<br />
• Changes in pH occur rapidly and, like temperature, is related to the time the culture<br />
vessel stays out <strong>of</strong> the incubator and the time <strong>of</strong> exposure to air.<br />
• Ensure that all surfaces are warmed and that all materials that may come in contact with<br />
the oocytes are sterile, non-toxic and <strong>of</strong> tissue culture quality, preferably mo<strong>use</strong> embryo<br />
tested.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
IVF-ET<br />
21
Insemination<br />
Hands on<br />
1 Transfer the oocytes to insemination dishes containing 75 000–200 000 sperm/mL and<br />
leave at +37°C and 6 % CO 2 over night.<br />
Alternatively: Add equilibrated sperm suspension to equilibrated dishes with the<br />
oocytes already present.<br />
Several oocytes may be inseminated in the same vessel. If oil overlay is <strong>use</strong>d, droplets <strong>of</strong><br />
at least 100 µL volume are recommended.<br />
Fertilisation assessment<br />
Essentials<br />
• IVF inseminated oocytes are checked for fertilisation approximately 15–20 hours after<br />
addition <strong>of</strong> sperm (day 1). ICSI oocytes may be observed 12–18 hours after injection,<br />
as pronuclei (PN) may appear earlier.<br />
• Fertilised oocytes are sensitive to temperature and pH variations and it is important to<br />
minimise these changes <strong>by</strong> using pre-warmed supplemented G-MOPS /G-MOPS <br />
PLUS as a handling medium outside <strong>of</strong> the incubator.<br />
• G-MOPS /G-MOPS PLUS contains the buffer MOPS to stabilise pH during handling<br />
in room atmosphere and does not require equilibration in a CO 2 atmosphere prior to<br />
<strong>use</strong>. Rather, this medium is designed to maintain a pH <strong>of</strong> 7.2 – 7.4 outside <strong>of</strong> a CO 2<br />
atmosphere.<br />
• G-GAMETE can be <strong>use</strong>d as an alternative to supplemented G-MOPS /G-MOPS <br />
PLUS. G-GAMETE should be equilibrated at +37°C and 6 % CO 2 before <strong>use</strong>.<br />
• It is recommended to heat all microscope stages and to work rapidly. To ensure that the<br />
temperature inside <strong>of</strong> the dish is correct, please verify <strong>by</strong> using a certified thermometer<br />
or a calibrated digital thermometer with a thermocouple that can be secured to the<br />
stage and to the inside <strong>of</strong> the dish.<br />
Hands on – fertilisation assessment<br />
1 Transfer the oocytes to a dish with pre-warmed supplemented G-MOPS /<br />
G-MOPS PLUS.<br />
2 Remove cumulus and corona cells from oocytes at +37°C using a denudation pipette,<br />
see Swemed Instruments <strong>by</strong> <strong>Vitrolife</strong> page 62. The cumulus and corona cells are <strong>of</strong>ten<br />
well dispersed <strong>by</strong> the hyaluronidase from the sperm. If the cumulus cells are not well<br />
22<br />
IVF-ET<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
dispersed, needles may be <strong>use</strong>d to attentively tease the oocyte out <strong>of</strong> the corona cells.<br />
Cells need only be removed to the extent that polar bodies and pronuclei can be clearly<br />
observed.<br />
3 Observe microscopically and record the number <strong>of</strong> pronuclei, polar bodies and the<br />
possible presence <strong>of</strong> a germinal vesicle. It is recommended to make these observations<br />
at high magnification (minimum 200X) on an inverted microscope with Nomarski<br />
or H<strong>of</strong>fman optics. It is difficult to be accurate in the assessment <strong>of</strong> fertilisation if lower<br />
magnifications are <strong>use</strong>d. Only embryos derived from normally fertilised oocytes (2 PN)<br />
should be considered for embryo culture and transfer. Non-fertilised or degenerated<br />
oocytes, oocytes with only 1 PN, and oocytes with more than 2 PN should be removed<br />
from culture. Pro-nuclear scoring, see the following page.<br />
4 After assessment, the fertilised oocytes should be rinsed thoroughly in several droplets<br />
<strong>of</strong> equilibrated supplemented G-1 /G-1 PLUS and then cultured in equilibrated supplemented<br />
G-1 /G-1 PLUS, preferably under oil.<br />
Scoring <strong>of</strong> human pronuclear embryos<br />
From: Scott L, Alvero R, Leondires M and Miller B. 2000. The morphology <strong>of</strong> human pronuclear embryos is<br />
positively related to blastocyst development and implantation. Hum Repr 15, No 11, pp 2394-2403<br />
Essentials<br />
• Zygote morphology has been related to blastocyst development, implantation and<br />
pregnancy<br />
• The scoring system is <strong>use</strong>d to assess the developmental capacity <strong>of</strong> zygotes and is<br />
based on the morphology <strong>of</strong> the two pronuclei (2PN)<br />
• Pronuclear scoring can be combined with scoring on Day 3 and/or blastocyst scoring<br />
• It is recommended to transfer not more than two embryos in order to avoid the<br />
complications <strong>of</strong> multiple pregnancies.<br />
Hands on<br />
• Scoring <strong>of</strong> zygotes should be performed on an inverted microscope while maintaining<br />
physiological pH and temperature.<br />
• Scoring should be performed 15-20 hours after insemination or 12-18 hours after<br />
ICSI, since the pronuclei <strong>of</strong> injected oocytes may appear as well as disappear earlier<br />
than for inseminated oocytes.<br />
• It is recommended to <strong>use</strong> supplemented G-MOPS /G-MOPS PLUS or<br />
G-GAMETE for this procedure.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 IVF-ET 23
SWIMUP D/E<br />
Scoring System<br />
• The nuclear size should be equal<br />
• The nucleoli need to be aligned at the pronuclear junction<br />
• The number <strong>of</strong> nucleoli should be between three and seven per nucleus with no more<br />
than one nucleolus difference between the nuclei<br />
• The nucleoli should be equal in size<br />
• To obtain optimal pregnancy and implantation rates, embryos <strong>of</strong> good morphological<br />
quality that showed pattern Z1 at the PN stage should preferably be chosen for transfer.<br />
Pattern DENS A GIII<br />
Z1 Z2 Z3 Z4<br />
A<br />
a)<br />
b)<br />
c)<br />
Choosing the culture system<br />
Z1 Z2 Z3 Z4<br />
DENS B,C,D GIII<br />
There are essentially two culture systems to choose from for culture <strong>of</strong> gametes and<br />
embryos: B C D<br />
A<br />
B<br />
Large volumes (0.5 mL – 1.0 mL) in tubes or wells, with or without oil<br />
Small volumes (droplets 0.05 mL – 0.1 mL) under oil<br />
a)<br />
The surface areas <strong>of</strong> systems without oil are large enough for the osmolality to change over<br />
several days. We therefore recommend the <strong>use</strong> <strong>of</strong> oil as a protection against changes in<br />
temperature and osmolality and as a barrier to dust particles and any microorganisms from<br />
the atmosphere. Paraffin oil is chosen for its viscosity and its high purity. OVOIL is pharmaceutical<br />
grade light paraffin oil, sterilised <strong>by</strong> filtration using a 0.22 µm pore size sterile<br />
PREPCULT A,B<br />
filter.<br />
A<br />
a)<br />
B<br />
a)<br />
b)<br />
A. Multi-well with OVOIL B. Droplets under oil<br />
a) OVOIL a) OVOIL<br />
b) Medium b) Medium droplets 10–100 µL<br />
MICRO A,B<br />
b)<br />
A a)<br />
24<br />
f)<br />
IVF-ET<br />
b)<br />
a) b)<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
c) d)
Preparation <strong>of</strong> culture system<br />
Embryo cultures should ideally be performed in 6 % CO 2 and 5 % O 2 for all media. This<br />
gas environment can be created using either a tri-gas incubator or a modular incubator<br />
chamber/isolette and fed with a gas mix (6 % CO 2 , 5 % O 2 and 89 % N 2 ). There are<br />
significant benefits to using a low oxygen environment. Should it not be possible to <strong>use</strong> a<br />
reduced oxygen environment, <strong>use</strong> 6 % CO 2 in air atmosphere.<br />
Hands on<br />
1 All culture dishes (wells or tubes) should be rinsed with G-RINSE and prepared in<br />
advance <strong>of</strong> the procedure.<br />
2 In the afternoon <strong>of</strong> the day <strong>of</strong> oocyte retrieval, label a micro-droplet dish with ID <strong>of</strong> the<br />
patient. Using a pre-rinsed sterile tip, fill an appropriate number <strong>of</strong> micro-wells up to<br />
25 µL <strong>of</strong> supplemented G-1/G-1 PLUS. Immediately cover the drops with OVOIL to<br />
avoid evaporation. Do not prepare more than 1 dish at the time.<br />
Alternatively, label a 40 mm culture dish with ID <strong>of</strong> the patient. Using a pre-rinsed sterile<br />
tip, place droplets <strong>of</strong> up to 50 µL <strong>of</strong> supplemented G-1/G-1 PLUS at the bottom <strong>of</strong><br />
the dish. Immediately cover the drops with OVOIL to avoid evaporation. Do not prepare<br />
more than 1 dish at the time.<br />
To achieve round standing droplets, you can do like this: place 6x25 µL droplets <strong>of</strong><br />
supplemented G-1/G-1 PLUS at the bottom <strong>of</strong> a 40 mm culture dish. Immediately<br />
cover the drops with OVOIL to avoid evaporation. Using a new tip for each drop, first<br />
rinse the tip and then take a further 25 µL <strong>of</strong> medium to each original drop. Do not prepare<br />
more than 1 dish at the time. This technique allows the droplets to stand up rather<br />
than flatten out.<br />
3 Immediately place the dish in the incubator at 6 % CO 2 and +37°C. Gently remove the<br />
lid <strong>of</strong> the dish and set at an angle on the side <strong>of</strong> the plate. This will ensure proper equilibration<br />
<strong>of</strong> the dish.<br />
Dishes must equilibrate in the incubator with a semi-opened lid for a minimum<br />
<strong>of</strong> 6 hrs (this is the minimal measured time for the media to reach correct pH<br />
under oil) and for a maximum <strong>of</strong> 18 hours.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 IVF-ET 25
Embryo culture<br />
Essentials<br />
• Crucial considerations for embryo culture are temperature, pH, and osmolality.<br />
• Temperature loss occurs rapidly and is related to the amount <strong>of</strong> time a culture vessel<br />
stays out <strong>of</strong> the incubator. It is recommended to work on heated surfaces, i.e. the<br />
microscope stage.<br />
• Changes in osmolality will occur slowly and is <strong>of</strong>ten an undetected parameter. The<br />
culture system should be correctly humidified and preferably under oil.<br />
• Changes in pH occur rapidly when medium is exposed to air. Ensure the culture dishes<br />
are out <strong>of</strong> the incubator for the minimal time period possible.<br />
• High implantation and pregnancy rates can be achieved <strong>by</strong> transfer <strong>of</strong> blastocysts.<br />
• Blastocyst culture and transfer will not rescue a deficient day 2 / day 3 IVF-ET program.<br />
• Cultivation <strong>of</strong> blastocysts for transfer and cryopreservation requires rigorous control <strong>of</strong><br />
laboratory conditions.<br />
• A sufficient number <strong>of</strong> incubators is a prerequisite for successful extended culture.<br />
Ideally, two incubators should be <strong>use</strong>d for no more than 5 retrievals per week. One <strong>of</strong><br />
the incubators should be <strong>use</strong>d for embryo culture, while the second should be <strong>use</strong>d for<br />
the pre-equilibration <strong>of</strong> the media dishes. Culture incubators should not be <strong>use</strong>d for preequilibration<br />
<strong>of</strong> media to minimize the number <strong>of</strong> door openings occurring.<br />
Hands on: Culture <strong>of</strong> Cleavage Stage Embryos<br />
Day 1 – Culture in G-1/G-1 PLUS<br />
1 Once the cumulus is removed, all manipulations should be performed using a pulled<br />
Pasteur pipette, see Swemed Instruments <strong>by</strong> <strong>Vitrolife</strong> page 62, Transfer pipette. It is<br />
important to <strong>use</strong> a pipette containing a tip with a diameter that is slightly larger than that<br />
<strong>of</strong> the embryo. To avoid damage, it is very important that the tip is not smaller than the<br />
embryo. For example, for embryos on day 1 to 3, a pipette tip <strong>of</strong> 150 to 200 µm would<br />
suffice. Using the appropriate size tip minimises the volumes <strong>of</strong> culture medium moved<br />
with each embryo, which typically should be less than one microlitre.<br />
Such attention to volume manipulation is a pre-requisite for successful culture.<br />
2 Following removal <strong>of</strong> the cumulus cells and assessment <strong>of</strong> fertilisation, pronucleate<br />
embryos are transferred to a centre well dish and washed in pre-warmed supplemented<br />
G-MOPS /G-MOPS PLUS or in equilibrated G-GAMETE . Washing entails picking<br />
up the embryo 2–3 times in a minimal volume and moving it around within the well.<br />
Embryos should then be washed successively in at least two drops in the culture dish.<br />
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More extensive washing will reduce the risk <strong>of</strong> transferring medium from the old dish to<br />
the fresh one. Residues <strong>of</strong> MOPS-buffer in the culture droplets may decrease pH below<br />
specification.<br />
3 Place the embryos in the equilibrated supplemented drops <strong>of</strong> G-1/G-1 PLUS. As<br />
a precautionary measure, prepare two culture dishes if the patient has more than 10<br />
embryos. Return the dish to the incubator immediately. It is advisable to culture embryos<br />
in at least groups <strong>of</strong> 2. For example, for a patient with 6 embryos it is best to culture in 2<br />
groups <strong>of</strong> 3 rather than 4 and 2 or 5 and 1.<br />
4 On day 3, embryos can be transferred to the uterus in equilibrated EmbryoGlue ® or in<br />
equilibrated supplemented G-2 /G-2 PLUS. Alternatively, on day 3, embryos can be<br />
transferred to equilibrated supplemented G-2 /G-2 PLUS for further culture to the<br />
blastocyst stage.<br />
Hands on: Culture <strong>of</strong> Blastocyst Stage Embryos<br />
Day 3 – Culture in G-2 /G-2 PLUS<br />
1 In the morning <strong>of</strong> day 3, label a micro-droplet dish with ID <strong>of</strong> the patient. Using a prerinsed<br />
sterile tip, fill an appropriate number <strong>of</strong> micro-wells up to 25 µL <strong>of</strong> supplemented<br />
G-2/G-2 PLUS. Immediately cover the drops with OVOIL to avoid evaporation. Do<br />
not prepare more than 1 dish at the time. Immediately place the dish in the incubator at<br />
6% CO 2 and +37°C. The minimum equilibration time is 6 hours before <strong>use</strong>. If the patient<br />
has more than 10 embryos make up two culture dishes.<br />
Alternatively, label a 40 mm culture dish with ID <strong>of</strong> the patient. Using a pre-rinsed sterile<br />
tip, place droplets <strong>of</strong> up to 50 µL <strong>of</strong> supplemented G-1/G-1 PLUS at the bottom <strong>of</strong><br />
the dish. Immediately cover the drops with OVOIL to avoid evaporation. Do not prepare<br />
more than 1 dish at the time.<br />
To achieve round standing droplets, you can do like this: place 9x25 µL droplets <strong>of</strong><br />
supplemented G-2/G-2 PLUS at the bottom <strong>of</strong> a 40 mm culture dish. Immediately<br />
cover the drops with OVOIL to avoid evaporation. Using a new tip for each drop, first<br />
rinse the tip and then take a further 25 µL <strong>of</strong> medium to each original drop. Do not prepare<br />
more than 1 dish at the time. This technique allows the droplets to stand up rather<br />
than flatten out. Immediately place the dish in the incubator at 6% CO2 and +37°C.<br />
The minimum equilibration time is 6 hours before <strong>use</strong>. If the patient has more than 10<br />
embryos make up two culture dishes.<br />
Alternatively, prepare the dishes for blastocyst culture in the afternoon <strong>of</strong> day 2 and<br />
equilibrate at 6 % CO 2 and +37°C over night.<br />
2 For each patient, set up one wash dish <strong>of</strong> pre-warmed supplemented G-MOPS /<br />
G-MOPS PLUS or equlibrated G-GAMETE per 10 embryos. Place 1 mL <strong>of</strong><br />
G-MOPS /G-MOPS PLUS/G-GAMETE into the well <strong>of</strong> a centre well dish. Place<br />
2 mL <strong>of</strong> G-MOPS /G-MOPS PLUS/G-GAMETE into the moat <strong>of</strong> the dish. Place on a<br />
heated stage.<br />
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3 For each patient, set up one sorting dish. Place 1 mL <strong>of</strong> G-MOPS /G-MOPS PLUS/<br />
G-GAMETE into the well <strong>of</strong> a centre well dish. Place 2 mL <strong>of</strong> G-MOPS /G-MOPS <br />
PLUS/G-GAMETE into the moat. Place on a heated stage.<br />
G-MOPS should not be placed in a CO 2 incubator, but rather pre-warmed in a<br />
sealed container using an incubator without CO 2 or a tube warming block.<br />
4 In the afternoon <strong>of</strong> day 3, the embryos that were assessed for cleavage in the morning<br />
are transferred to equilibrated supplemented G-2 /G-2 PLUS. The embryos will<br />
remain in the supplemented G-2 /G-2 PLUS in 6 % CO 2 and +37°C until the assessment<br />
for transfer on the morning <strong>of</strong> day 5. If there is concern about the embryo cleavage<br />
rate, nursing and medical staff should be notified.<br />
5 Wash embryos in the wash dish (this step is crucial to remove the EDTA in G-1 /<br />
G-1 PLUS). Washing entails picking up the embryo in a minimal volume <strong>of</strong> media 2–3<br />
times and moving it around within the well. After washing, transfer embryos to the sorting<br />
dish and group like embryos together. Rinse through the wash drops <strong>of</strong> the equilibrated<br />
supplemented G-2 /G-2 PLUS in the culture dish and again place up to five<br />
embryos in each <strong>of</strong> the culture drops. Return the dish to the CO 2 incubator immediately.<br />
6 On day 4, prepare three dishes <strong>of</strong> supplemented G-2 /G-2 PLUS. Label the dishes<br />
“Transfer”, “Freeze” and “Hold” (for culture to day 6). The dishes should be equilibrated<br />
overnight at +37°C and 6 % CO 2 . Dishes should be pre-equilibrated for no less than 6<br />
hours prior to <strong>use</strong>.<br />
7 Transfers should be performed either in equilibrated EmbryoGlue ® or in equilibrated<br />
supplemented G-2 /G-2 PLUS. Equilibrate transfer dishes at +37°C and 6 % CO 2 at<br />
least 6 hours prior to <strong>use</strong>.<br />
8 On day 5, embryos should be evaluated and one or two top scoring blastocysts selected<br />
for transfer. Good quality blastocysts not transferred can be cryopreserved. Should<br />
an embryo not have formed a blastocyst <strong>by</strong> day 5, it should be cultured in a fresh drop <strong>of</strong><br />
equilibrated supplemented G-2 /G-2 PLUS for 24 hours and assessed on day 6.<br />
9 Move the selected blastocyst(s) to supplemented G-2 /G-2 PLUS and leave at +37°C<br />
and 6 % CO 2 until 10–30 minutes before transfer.<br />
Embryo assessment<br />
Essentials<br />
• Embryos may be transferred to the uterus on day 2 (40–48 hours post insemination)<br />
or day 3 (66–74 hours post insemination), or at the blastocyst stage on day 5–6<br />
(120–144 hours post insemination).<br />
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• The embryo morphology is assessed as close to the time <strong>of</strong> transfer as possible.<br />
• The early embryos are assessed for total number <strong>of</strong> blastomeres, regularity <strong>of</strong> the<br />
blastomeres, presence and percentage <strong>of</strong> fragmentation, granularity <strong>of</strong> blastomeres,<br />
and cleavage rate. For assessment <strong>of</strong> blastocysts, see “Scoring criteria for human<br />
blastocysts” on the following pages.<br />
• The embryo(s) chosen for transfer are placed into a fresh dish <strong>of</strong> equilibrated<br />
EmbryoGlue ® or equilibrated supplemented G-2 /G-2 PLUS, irrespective <strong>of</strong> stage.<br />
Scoring criteria for human blastocysts<br />
From: <strong>Gardner</strong> DK and Schoolcraft WB (1999) In-vitro culture <strong>of</strong> human blastocysts. in Towards<br />
Reproductive Certainty: Infertility and Genetics Beyond 1999. eds. Jansen, R. and Mortimer, D.<br />
Parthenon Press, Carnforth, pp 378-388.<br />
Essentials<br />
• The scoring system is <strong>use</strong>d to assess the developmental capacity <strong>of</strong> blastocysts<br />
based on their morphological appearance, and there<strong>by</strong> enable selection for transfer or<br />
cryopreservation.<br />
• Scoring <strong>of</strong> blastocysts should be performed on an inverted microscope while<br />
maintaining physiological pH and temperature.<br />
• The majority <strong>of</strong> the blastocysts should develop <strong>by</strong> day 5 (see below).<br />
• The blastocysts selected for transfer (maximum two) should be placed in the dish<br />
labeled “Transfer”, and the blastocysts selected for cryopreservation should be placed<br />
in the dish labeled “Freeze”.<br />
• It is recommended to transfer not more than one blastocyst in order to avoid the<br />
complications <strong>of</strong> multiple pregnancies. Preference should be given to blastocysts<br />
graded as 3 and higher. Select the best scoring blastocysts preferentially, i.e. AA.<br />
Hands on<br />
1 Blastocysts are given an alphanumeric score from 1 to 6, based on their degree <strong>of</strong><br />
expansion and hatching status and two letter scores for inner cell mass (ICM) and trophectoderm.<br />
2 The initial phase <strong>of</strong> the assessment can be performed on a dissection microscope.<br />
3 The second step in scoring the blastocysts should be performed on an inverted microscope.<br />
For blastocysts graded as 3 to 6 (i.e. full blastocysts onwards) the development<br />
<strong>of</strong> the inner cell mass (ICM) and trophectoderm can then be assessed.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 IVF-ET 29
Scoring system<br />
Degree <strong>of</strong> expansion and hatching status:<br />
1 2 3 4<br />
1 Early blastocysts the blastocoel being less than half the volume <strong>of</strong> the embryo.<br />
2 Blastocyst the blastocoel being greater than or equal to half <strong>of</strong> the<br />
volume <strong>of</strong> the embryo.<br />
3 Full blastocyst the blastocoel completely fills the embryo.<br />
4 Expanded blastocyst the blastocoel volume is now larger than that <strong>of</strong> the early<br />
embryo and the zona is thinning.<br />
5 Hatching blastocyst the trophectoderm has started to herniate through the zona.<br />
6 Hatched blastocyst the blastocyst has completely escaped from the zona.<br />
Inner cell mass (ICM) Grading:<br />
A<br />
B<br />
C<br />
Tightly packed, many cells<br />
Loosely grouped, several cells<br />
Very few cells<br />
Trophectoderm Grading:<br />
A<br />
B<br />
C<br />
Many cells forming a cohesive epithelium<br />
Few cells forming a loose epithelium.<br />
Very few cells<br />
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Example <strong>of</strong> a 4AA blastocyst.<br />
Focus on: Inner cell mass<br />
Focus on: Trophectoderm<br />
Table for assessment <strong>of</strong> blastocysts<br />
Degree <strong>of</strong> Inner cell mass Trophectoderm grading<br />
expansion/<br />
hatching Tightly Loosely Very few Many cells Few cells Very few<br />
status packed, grouped, cells forming a forming a cells<br />
many cells several cells cohesive loose<br />
epithelium epithelium<br />
3 Full blastocyst;<br />
the blastocoel<br />
completely fills<br />
the embryo<br />
4 Expanded<br />
blastocyst; the<br />
blastocoel volume<br />
is now larger than<br />
that <strong>of</strong> the early<br />
embryo and the<br />
zona is thinning<br />
5 Hatching<br />
blastocyst; the<br />
trophectoderm<br />
has started to<br />
herniated through<br />
the zona<br />
6 Hatched<br />
blastocyst; the<br />
blastocyst has<br />
completely<br />
escaped from<br />
the zona<br />
A B C A B C<br />
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Blastocyst schedule (an example)<br />
The blastocyst culture schedule is quite different from the routine IVF day 2 / day 3<br />
schedule. Here is a day-to-day summary.<br />
We suggest that you make this page visible to all laboratory personnel.<br />
If PLUS products are <strong>use</strong>d no supplementation will be needed. Otherwise, supplement<br />
all media with either G-MM or HSA-solution , except EmbryoGlue ® , G-GAMETE and<br />
G-RINSE .<br />
Day –1<br />
(the day before<br />
oocyte collection)<br />
Prepare G-RINSE,<br />
G-MOPS and G-IVF<br />
for oocyte collection and<br />
insemination as for routine<br />
IVF protocol. For ICSI<br />
prepare also G-1 dishes.<br />
If G-GAMETE is <strong>use</strong>d,<br />
prepare dishes for oocyte<br />
wash.<br />
Day 0<br />
Oocyte collection and<br />
insemination in G-IVF. ICSI<br />
oocytes should be placed<br />
in G-1 immediately after<br />
injection. Prepare G-1<br />
dishes late in the afternoon<br />
for <strong>use</strong> Day‐1. Prepare<br />
G-MOPS dishes for oocyte<br />
retrieval.<br />
Day 1<br />
Assess IVF and ICSI<br />
fertilisation. Rinse in<br />
G-MOPS/G-GAMETE<br />
and place into G-1 for<br />
culture.<br />
Day 2<br />
Embryo assessment optional.<br />
Day 3<br />
Assess embryos for cleavage.<br />
Rinse embryos in G-MOPS/<br />
G-GAMETE and wash<br />
well and transfer to G-2 for<br />
culture, early afternoon.<br />
Day 4<br />
Embryo assessment<br />
optional. Prepare dishes with<br />
EmbryoGlue® or G-2 for<br />
transfer and G-2 dishes for<br />
freezing and hold to day 6,<br />
late in the afternoon.<br />
Day 5<br />
Assess morphology <strong>of</strong><br />
blastocysts. Use scoring<br />
criteria for transfer, freezing or<br />
hold. Transfer blastocysts to<br />
EmbryoGlue® or fresh G-2<br />
before transfer to the uterus.<br />
Day 6<br />
Assess morphology <strong>of</strong><br />
blastocysts. Use scoring<br />
criteria for transfer or freezing.<br />
Blastocysts scoring less than<br />
3BB or below <strong>by</strong> day 6, do not<br />
cryopreserve very well. Transfer<br />
blastocysts to EmbryoGlue®<br />
or fresh G-2 before transfer<br />
to the uterus.<br />
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Embryo transfer<br />
Embryo transfer (ET) is the process <strong>of</strong> placing the embryos into the uterus. It is usually performed<br />
transcervically, without anaesthesia.<br />
Essentials<br />
• Sterile non-toxic non-powdered gloves should be worn when handling the catheter.<br />
• Great care should be taken to avoid contamination <strong>of</strong> the catheter tip at all times.<br />
• It is common practice to transfer one or two embryos per patient.<br />
• A large volume (60 microliters) <strong>of</strong> transfer media and a large air interface may result<br />
in expulsion <strong>of</strong> embryos into the cervix or to the outside <strong>of</strong> the catheter. Removing<br />
the air column can minimise such complications. Therefore it is recommended that a<br />
continuous fluid column <strong>of</strong> approximately 30 microliters <strong>of</strong> equilibrated EmbryoGlue ®<br />
or equilibrated supplemented G-2 /G-2 PLUS is <strong>use</strong>d in a catheter attached to a 1cc<br />
airtight syringe. Load the embryos preferentially toward the tip <strong>of</strong> the column <strong>of</strong> transfer<br />
media, closest to the catheter opening.<br />
Replacement <strong>of</strong> embryos<br />
Hands on for the gynaecologist<br />
1 Arrange in advance for the patient to arrive for the ET with a partially full bladder. In most<br />
cases this helps to position the uterus for easy entry with a s<strong>of</strong>t, atraumatic catheter. In<br />
the situation <strong>of</strong> a retroverted uterus, the patient may empty her bladder.<br />
2 A non-toxic, quality tested, single <strong>use</strong>, atraumatic catheter must be <strong>use</strong>d.<br />
3 It is recommended to perform embryo transfer under ultrasound guidance.<br />
4 Place the patient into lithotomy position. Expose the cervix with the <strong>use</strong> <strong>of</strong> a speculum<br />
washed with equilibrated G-RINSE with added antibiotics. It may be necessary to<br />
remove the cervical mucus with a sterile syringe.<br />
5 An ultrasound scan during the ET (abdominally) is highly recommended for guidance <strong>of</strong><br />
the catheter in its passage through the cervical canal. A trial catheter may be introduced<br />
to test the ease <strong>of</strong> passage. If the passage is easy, the embryos may be loaded into a<br />
clean catheter and the ET performed. If the trial catheter meets some obstruction several<br />
options may be considered. An obturator with some flexibility may be <strong>use</strong>d to direct<br />
the catheter into the cavity. Whichever is <strong>use</strong>d, attention should be paid to minimise<br />
trauma to the cervical canal and cavity.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 IVF-ET 33
6 The operator performing the ET should be careful not to expel the embryos from the<br />
catheter <strong>by</strong> bending, squeezing or causing damage to it.<br />
7 The catheter should pass through the cervical canal and beyond the inner os. Tactile<br />
feedback may be sufficient for the operator to accomplish this. The catheter should be<br />
clearly marked so that it can be determined how far it has been introduced. Ultrasound<br />
should be <strong>use</strong>d to confirm the position.<br />
8 Expel the embryo(s) into the uterus in approximately 30 µL <strong>of</strong> transfer medium and<br />
slowly withdraw the catheter while maintaining steady pressure on the plunger <strong>of</strong> the<br />
syringe.<br />
9 Make a final microscopic examination <strong>of</strong> the catheter.<br />
For a more complete review <strong>of</strong> this topic see: Schoolcraft, Surrey and <strong>Gardner</strong><br />
(2001) Embryo transfer: techniques and variables affecting success.<br />
Fertil. Steril.76; 863-870.<br />
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Loading the catheter<br />
Hands on for the laboratory<br />
1 Embryo transfer can be performed either in equilibrated EmbryoGlue ® or in equilibrated<br />
supplemented G-2 /G-2 PLUS.<br />
2 Add approximately 1 mL <strong>of</strong> EmbryoGlue ® to the well <strong>of</strong> a rinsed centre well dish.<br />
3 Add approximately 2 mL <strong>of</strong> EmbryoGlue ® to the moat <strong>of</strong> the centre well dish.<br />
4 Pre-equilibrate the dish in +37°C and 6 % CO 2 for 4–18 hours.<br />
5 Pre-equilibrate the embryos to be transferred in the well containing EmbryoGlue ® for<br />
a minimum <strong>of</strong> 10 minutes in a 6% CO 2 environment prior to transfer. Embryos can<br />
be held in EmbryoGlue for up to 4 hours. Embryos should not be kept in EmbryoGlue<br />
overnight.<br />
6 Rinse the 1 mL non-toxic syringe <strong>by</strong> drawing up and then out media from the moat<br />
several times until no air bubbles are observed in the syringe. Draw up approximately<br />
0.5 mL <strong>of</strong> the medium from the moat.<br />
7 Firmly attach the transfer catheter to the pre-rinsed 1 mL non-toxic syringe. Flush<br />
approximately 0.5–1.0 mL <strong>of</strong> equilibrated transfer medium from the moat through and<br />
out <strong>of</strong> the catheter.<br />
8 After rinsing, draw approximately 0.1 mL <strong>of</strong> EmbryoGlue ® from the center well and expel<br />
into the moat until approximately 20 µL is left in the syringe.<br />
9 Under microscopic control, gently load the embryos into the catheter in approximately<br />
5–10 µL <strong>of</strong> additional EmbryoGlue ® followed <strong>by</strong> a small amount <strong>of</strong> air. (The small pocket<br />
<strong>of</strong> air at the tip allows better imaging <strong>of</strong> the tip for ultrasound guided transfers). For the<br />
embryo transfer, pass the tip <strong>of</strong> the catheter into the uterus approximately 1 cm from the<br />
top <strong>of</strong> the cavity and expel the embryos in a total volume <strong>of</strong> approximately 25–30 µL <strong>of</strong><br />
medium. Slowly withdraw the catheter while maintaining steady pressure on the plunger<br />
<strong>of</strong> the syringe. Make a final microscopic examination <strong>of</strong> the catheter.<br />
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Micro<br />
Manipulation<br />
Micromanipulation includes the procedures <strong>of</strong> ICSI (Intra Cytoplasmic Sperm Injection),<br />
assisted hatching and embryo biopsy.<br />
Essentials<br />
• All micromanipulation equipment should be correctly aligned and positioned for<br />
maximum stability. It is recommended to keep it separate from vibrations ca<strong>use</strong>d <strong>by</strong><br />
doors, elevators, air currents and through traffic that may ca<strong>use</strong> stressful interruption<br />
and noise.<br />
• The inverted microscope should have a heated stage correctly adjusted to maintain fluid<br />
in the dish at +37°C. Temperature maintenance during the procedures is important.<br />
• Osmolality is controlled <strong>by</strong> using droplets under equilibrated oil.<br />
• Highest quality non-toxic micro-tools should be <strong>use</strong>d for all procedures for consistency<br />
and quality control, see Swemed Instruments <strong>by</strong> <strong>Vitrolife</strong> page 62, ICSI- and Holding<br />
Pipettes.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
Micro Manipulation<br />
37
Denudation <strong>of</strong> oocytes for ICSI<br />
If ICSI is to be performed, the oocytes will need to have their cumulus mass and corona<br />
removed. DENS This A GIII process is called denudation.<br />
This process may be performed either using the large volume method without oil, using<br />
A<br />
multi-wells and dishes or the droplet method under oil.<br />
HYASE (hyaluronidase) is <strong>use</strong>d to facilitate the dispersal <strong>of</strong> the cumulus mass and corona.<br />
Using fine<br />
a)<br />
glass pipettes, see Swemed Instruments <strong>by</strong> <strong>Vitrolife</strong> page 62, Denudation<br />
pipettes, b) cells are removed <strong>by</strong> gently pipetting the oocyte up and down. The diameter <strong>of</strong> the<br />
pipette<br />
c)<br />
should be slightly larger than that <strong>of</strong> the oocyte (approximately 130–175 µm). When<br />
using glass pipettes, several different sized pipettes may be needed. Using a pipette that is<br />
too narrow or has a jagged edge may damage the oocyte. Exposure to HYASE for too long<br />
or rough handling as well as exposure to sub-physiological pH and temperatures, may also<br />
damage DENS the B,C,D oocyte. GIII Oocytes in the germinal vesicle stage are particularly sensitive. For the<br />
above reasons it is important to <strong>use</strong> the correct concentration <strong>of</strong> HYASE and to keep to the<br />
recommended exposure time. HYASE is concentrated 10 times and should be diluted 1:10<br />
B C D<br />
with supplemented G-MOPS /G-MOPS PLUS or with G-GAMETE .<br />
CAUTION<br />
Exposure to HYASE for longer than 30 seconds may damage the oocyte.<br />
a)<br />
Hands on<br />
PREPCULT A,B<br />
1 Dilute HYASE with supplemented G-MOPS/G-MOPS PLUS or with<br />
G-GAMETE.<br />
A<br />
a)<br />
2 Prepare rinsed denudation dishes with diluted HYASE and supplemented<br />
G-MOPS/G-GAMETE for oocyte wash.<br />
B<br />
3 For every well <strong>of</strong> b) diluted HYASE , prepare four washing b) wells <strong>of</strong> approximately 1 mL or<br />
6 droplets (50–100 µL) <strong>of</strong> supplemented G-MOPS /G-MOPS PLUS/G-GAMETE <br />
under oil (A, B). Equilibrate at +37°C at room atmosphere for approximately 15 minutes,<br />
if G-MOPS/G-MOPS PLUS is <strong>use</strong>d. If G-GAMETE is <strong>use</strong>d, dishes should be<br />
MICRO A,B<br />
equilibrated in 6% CO 2 until correct pH has been attained, preferrably over night. At the<br />
same time prepare ICSI dishes (see below “HANDS ON - SET UP”).<br />
A<br />
f)<br />
a)<br />
b)<br />
a)<br />
a) b)<br />
38<br />
c) d)<br />
e)<br />
c)<br />
d)<br />
A. BDroplets under oil.<br />
a) HYASE solution b –f) Supplemented G-MOPS/G-MOPS PLUS/G-GAMETE wash droplets<br />
b)<br />
a)<br />
Micro Manipulation<br />
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d)<br />
c)
B<br />
b)<br />
a)<br />
e)<br />
c)<br />
d)<br />
B. 5-well dish. With or without oil.<br />
a) HYASE solution<br />
b–e) Supplemented G-MOPS/G-MOPS PLUS/G-GAMETE, wash droplets<br />
4 Using a large bore pipette, see Swemed Instruments <strong>by</strong> <strong>Vitrolife</strong> page 62, Transfer<br />
pipette, place 3–5 oocytes into the diluted HYASE . Gently pipette the hyaluronidase<br />
and the oocytes. The cumulus cells will start to disperse. It is important not to expose<br />
the oocytes to the hyaluronidase solution for longer than 30 seconds.<br />
5 Move the partly denuded oocytes into the first wash volume and take care to carry over<br />
a minimum amount <strong>of</strong> hyaluronidase solution. Aspirate each oocyte singly up and down,<br />
using a fine bore denudation pipette, see Swemed Instruments <strong>by</strong> <strong>Vitrolife</strong> page 62,<br />
Denudation pipette, to remove the corona. Rinse the oocytes in warmed supplemented<br />
G-MOPS /G-MOPS PLUS or in equilibrated G-GAMETE . Repeat with new dishes<br />
until all oocytes are denuded.<br />
6 Observe the oocytes for maturity <strong>by</strong> examining the presence (M2) or absence (M1) <strong>of</strong><br />
a polar body or presence <strong>of</strong> a germinal vesicle (GV). Place all mature oocytes (M2) into<br />
the prepared ICSI droplets, if the oocytes are to be injected immediately. If the denuded<br />
oocytes are to be incubated for some time before injection, the oocytes should be<br />
placed in supplemented G-1 /G-1 PLUS until the time <strong>of</strong> injection. Immature oocytes<br />
(M1 and GV) may be placed in culture medium for further incubation and maturation.<br />
Hands on - set up<br />
1 Pre-warm supplemented G-MOPS /G-MOPS PLUS and OVOIL at +37°C for<br />
approximately 15 minutes, or equilibrate G-GAMETE at +37°C and 6 % CO 2 for at<br />
least 4 hours.<br />
2 Remove ICSI (viscous sperm injection solution) from cold storage and equilibrate to a<br />
temperature <strong>of</strong> +20 ± 5°C.<br />
3 Dishes for ICSI should be made quickly. Place a 1–10 µL drop <strong>of</strong> ICSI in the center <strong>of</strong><br />
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Micro Manipulation<br />
39
a)<br />
c)<br />
d)<br />
an ICSI-dish and 6–10 µL droplets <strong>of</strong> supplemented G-MOPS/G-MOPS PLUS/G-<br />
GAMETE, cover with OVOIL. Do not prepare more than one dish at a time to avoid<br />
evaporation in the droplets. Work rapidly and have all items needed close at hand (A, B).<br />
MICRO Make up two SETUP dishes A,B per patient.<br />
A<br />
1<br />
B<br />
a)<br />
6 2<br />
1<br />
4<br />
b)<br />
2<br />
5<br />
a)<br />
5<br />
3<br />
3<br />
6<br />
4<br />
A. Option 1 B. Option 2<br />
a) Oocyte droplets, 1 to 6 a) Oocyte droplets, 1 to 6<br />
(G-MOPS PLUS/G-GAMETE)<br />
(G-MOPS PLUS/G-GAMETE)<br />
b) ICSI b) ICSI<br />
LOADSTRAW CRYO 1<br />
b)<br />
4 Warm the dishes at +37°C for at least 15 minutes if G-MOPS PLUS is <strong>use</strong>d.<br />
If G-GAMETE is <strong>use</strong>d, equilibrate the dishes at +37°C and 6 % CO 2 for at least 4 hours.<br />
a) b) c) d) e) f) g) h) i)<br />
ICSI procedure<br />
a) Syringe b) Plastic tube c) Cotton plug sealing cement d) FS 2, 2cm e) Air bubble 1/4 cm<br />
f) FS 2 + embryos, 2–3 cm g) Air bubble 1/4 cm h) FS 2, 1 cm i) Seal: plastic plug<br />
ICSI is the procedure <strong>of</strong> injecting the sperm into the oocyte. ICSI enables pregnancies to<br />
be achieved in couples with severe male factor infertility.<br />
LOADSTRAW CRYO 2<br />
Polyvinylpyrrolidone (PVP) is commonly <strong>use</strong>d in ICSI beca<strong>use</strong> <strong>of</strong> its viscous properties. It<br />
slows the motility <strong>of</strong> the sperm and makes it easier to “catch” the sperm, allowing correct<br />
immobilisation <strong>of</strong> the sperm and “crushing” <strong>of</strong> the tail membrane before injection. It also<br />
a) b) c) d) e) f) g) h) i)<br />
allows for controlled injection <strong>of</strong> the sperm <strong>by</strong> slowing down the speed <strong>of</strong> the injection fluid.<br />
This helps to stabilise the microinjection procedure and minimise the volume <strong>of</strong> fluid that is<br />
injected into the oocyte. All these factors contribute to success <strong>of</strong> the procedure.<br />
Essentials<br />
a) Syringe b) Plastic tube c) Cotton plug sealing cement d) BFS 2, 2cm e) Air bubble 1/4 cm<br />
f) BFS 2 + embryos, 2–3 cm g) Air bubble 1/4 cm h) BFS 2, 1 cm i) Seal: plastic plug<br />
• Immobilise the sperm correctly <strong>by</strong> swiping just below the neck <strong>of</strong> the sperm with the<br />
injection pipette (“crush tail”).<br />
• Inject sperm deep (50–75 % <strong>of</strong> the oocyte diameter) into the cytoplasm and ensure the<br />
sperm is not dragged out with the injection pipette.<br />
40<br />
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<strong>Vitrolife</strong> G-Series Manual 6.0
• To avoid the expulsion <strong>of</strong> the sperm into the perivitelline space, be sure the oolemma is<br />
broken <strong>by</strong> gentle suction <strong>of</strong> a small amount <strong>of</strong> cytoplasm.<br />
• Minimise the volume <strong>of</strong> ICSI (PVP) injected into the oocyte.<br />
For ICSI and Holding pipettes, see Swemed Instruments <strong>by</strong> <strong>Vitrolife</strong> page 62.<br />
Hands on<br />
1 Place a small volume (1–2 µL) <strong>of</strong> prepared sperm suspension into the centre <strong>of</strong> the<br />
ICSI droplet. Warm the dishes for a few minutes on a heated stage to allow migration<br />
<strong>of</strong> sperm to the outer perimeter <strong>of</strong> the droplet.<br />
2 Prime the injection pipette with ICSI to reduce the risk <strong>of</strong> the sperm sticking to the<br />
inside <strong>of</strong> the pipette.<br />
3 Place denuded oocytes into droplets <strong>of</strong> supplemented supplemented G-MOPS /G-<br />
MOPS PLUS/G-GAMETE , one oocyte per droplet, maximum 4 oocytes at a time.<br />
4 Immobilise individual sperm <strong>by</strong> using the injection pipette to “crush” the membrane<br />
<strong>of</strong> the sperm tail. It is important not to damage the neck region <strong>of</strong> the sperm since it<br />
contains the centriole, which is crucial for the migration <strong>of</strong> chromosomes at cell division.<br />
Inefficient tail “crushing” will lead to reduced fertilisation rates. Aspirate the individual<br />
immobilised sperm.<br />
5 Move the oocyte droplet into the field <strong>of</strong> view. Use a holding pipette to secure the<br />
oocyte for injection. Position the oocyte with the polar body at the 7 or 1 o’clock position.<br />
For minimal risk <strong>of</strong> damaging the meiotic spindle, inject the sperm at the 4‐o’clock<br />
position. Inject the sperm with the least amount <strong>of</strong> ICSI possible and after injection,<br />
make sure to stop the flow in the injection pipette. Pay attention to the time the dishes<br />
are outside <strong>of</strong> the incubator. Fewer oocytes per dish may be set up when the operator is<br />
inexperienced or the case is difficult.<br />
6 After all oocytes are injected, rinse through several drops <strong>of</strong> supplemented G-1 /<br />
G-1 PLUS and then place into prepared G-1 culture systems for overnight culture.<br />
7 The oocytes are assessed for fertilisation <strong>by</strong> the presence <strong>of</strong> two PN and two polar<br />
bodies on the following day. Non-fertilised, degenerated oocytes and oocytes with<br />
more than 2 PN or less than 2 PN should be removed from culture. After assessment,<br />
the zygotes should be rinsed in equilibrated supplemented G-1 /G-1 PLUS and then<br />
transferred to a new dish with equilibrated supplemented G-1 /G-1 PLUS for culture.<br />
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Micro Manipulation<br />
41
Difficult ICSI cases<br />
The level <strong>of</strong> difficulty <strong>of</strong> a case is mainly determined <strong>by</strong> three factors:<br />
• Operator<br />
• Equipment<br />
• Sperm sample<br />
The training, experience, endurance, and ability <strong>of</strong> the operator determine the first factor. It is very<br />
important when setting up ICSI that sufficient time and resources are available, particularly for difficult<br />
cases. The equipment should be easy to <strong>use</strong> and provide a stable setting to enable the operator<br />
to concentrate on the difficulties encountered in trying to find and pick the sperm. An ergonomically<br />
designed ICSI workstation allows the work to progress smoother and increases the endurance <strong>of</strong><br />
the operator.<br />
The sperm sample can also present difficulties. For example, sperm suspensions prepared from testicular<br />
biopsies as well as severe cases <strong>of</strong> oligoasthenoteratospermia usually exhibit very low count,<br />
low and sluggish motility, and presence <strong>of</strong> debris and other cells. Frozen thawed epididymal sperm<br />
may also be problematic. Only a small fraction <strong>of</strong> the sperm may survive and the surviving sperm may<br />
exhibit a very low progressive movement or no movement at all.<br />
A third example <strong>of</strong> a difficult ICSI case is sperm obtained <strong>by</strong> Electro-ejaculation. In this case, the<br />
sperm count can be very high and the motility very low. In addition there are other cells and debris<br />
present.<br />
Low sperm count, motility and progression<br />
Use SpermGrad to retrieve sperm (see Sperm Preparation). Add the sperm suspension to<br />
a droplet <strong>of</strong> equilibrated supplemented G-IVF /G-IVF PLUS. Transfer the sperm to ICSI <br />
before proceeding.<br />
Note that in cases <strong>of</strong> extremely low sperm count, the semen sample can be washed in<br />
equilibrated supplemented G-IVF /G-IVF PLUS and centrifuged at 300–600 g for 10<br />
minutes without previous gradient centrifugation. Re-suspend the spermatozoa in<br />
200–500 µL <strong>of</strong> equilibrated supplemented G-IVF /G-IVF PLUS and <strong>use</strong> as soon as<br />
possible.<br />
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<strong>Vitrolife</strong> G-Series Manual 6.0
Testicular biopsy<br />
Use G-MOPS PLUS to collect the sample. Transfer the tissue to a sterile non toxic Petri<br />
dish and disperse the material into small pieces.<br />
Transfer the dispersed material to small conical test tubes and centrifuge for 5 minutes<br />
at 300-600g. Discard the supernatants and re-suspend the pellets in G-MOPS PLUS.<br />
A second wash may be necessary.<br />
Place 5-10μL droplets <strong>of</strong> the testicular material in a small Petri dish and cover with OVOIL .<br />
Retrieve the testicular spermatozoa and collect in a separate clean droplet <strong>of</strong><br />
G-MOPS PLUS until injection.<br />
If the preparations are to be <strong>use</strong>d the following day, the material should be incubated in supplemented<br />
G-IVF /G-IVF PLUS at +37˚C and 6% CO 2 .<br />
Embryo biopsy<br />
Embryo biopsy is the procedure <strong>of</strong> removing a blastomere from the embryo for<br />
Pre-implantation Genetic Diagnosis (PGD).<br />
Essentials<br />
• Embryo biopsy is usually performed on the morning <strong>of</strong> day 3, before compaction <strong>of</strong> the<br />
blastomeres begins.<br />
• A hole in the zona can be drilled either with Tyrode’s acid, a laser or <strong>by</strong> partial zona<br />
dissection.<br />
• A separate pipette is needed to remove the biopsied blastomere. See Swemed<br />
Instruments <strong>by</strong> <strong>Vitrolife</strong> page 62, Blastomere Biopsy Pipette.<br />
CAUTION<br />
G-PGD is MOPS buffered and must be handled exactly like G-MOPS (see<br />
page 8). G-PGD must never be placed in a CO 2 incubator, but rather warmed in<br />
a tube warming block or in an incubator without CO 2 .<br />
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Micro Manipulation<br />
43
Hands on<br />
1 Prepare dishes with droplets <strong>of</strong> pre-warmed supplemented G-PGD covered with<br />
OVOIL .<br />
2 Wash the embryo in several droplets <strong>of</strong> supplemented G-PGD to remove the culture<br />
medium.<br />
3 Place the embryo in G-PGD (MOPS buffered, Ca 2+ - and Mg 2+ -free medium) at +37°C.<br />
4 Secure the embryo with the holding pipette.<br />
5 Drill a hole in the zona. Introduce a clean biopsy pipette into the hole and gently aspirate<br />
one or two blastomeres for analysis.<br />
6 After extensive washing in G-2 /G-2 PLUS, to remove G-PGD , place the embryos<br />
into equilibrated supplemented G-2 /G-2 PLUS for culture until the results <strong>of</strong> the<br />
embryo biopsy analysis are confirmed. The non-affected embryos may be selected for<br />
fresh embryo transfer on day 5, or cryopreservation if there is a surplus. The embryo<br />
transfer is usually performed on day 5, after the genetic diagnosis has been completed.<br />
CAUTION<br />
• Spend time adjusting the embryo position on the holding pipette so that a<br />
nucleated blastomere can be clearly identified and biopsied.<br />
• If compaction has occurred, there will be junctions between the blastomeres<br />
and they may be more difficult to remove. Be patient, pull and move the<br />
pipette across and back to ease the blastomere out.<br />
• Jagged edges on the biopsy pipette will ca<strong>use</strong> the biopsied cell to lyse.<br />
Therefore, only <strong>use</strong> the highest quality pipettes.<br />
44 <strong>Vitrolife</strong> G-Series Manual 6.0
Embryo<br />
Cryopreservation<br />
Essentials<br />
• The results <strong>of</strong> cryopreservation are dependent on the baseline IVF success rate <strong>of</strong> the<br />
clinic. If the fresh IVF results are low, this will also be reflected in the cryopreservation<br />
results.<br />
• The results are dependent on well maintained and accurately calibrated freezing<br />
equipment, careful handling and detailed attention to choosing only embryos with good<br />
morphological appearance.<br />
Equipment list<br />
This is a suggested list and alternatives may exist.<br />
1 Cryopreservation machine: Base your purchase decision on cost, available space and<br />
ability for maintenance and service.<br />
2 Storage vessel: This should be a vessel <strong>use</strong>d only for human embryos. Liquid nitrogen<br />
submersion is the traditional method. Separate vessels should be <strong>use</strong>d if you cryopreserve<br />
embryos from patients with infectious diseases.<br />
3 Straws or plastic cryo-vials for freezing: These need to be sterile, non-toxic and <strong>of</strong> high<br />
quality.<br />
4 The amount <strong>of</strong> liquid nitrogen (LN 2 ) <strong>use</strong>d is dependent upon the choice <strong>of</strong> equipment<br />
and storage system.<br />
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Embryo Cryopreservation<br />
45
Cryopreservation is the freezing and thawing <strong>of</strong> cells with minimal damage. This is achieved <strong>by</strong> the<br />
<strong>use</strong> <strong>of</strong> a cryoprotectant that dehydrates the cells and disturbs crystallisation <strong>of</strong> water during cooling.<br />
Selecting an appropriate temperature pr<strong>of</strong>ile during cooling further facilitates this process so that<br />
the cells become maximally dehydrated. Introduction <strong>of</strong> cryoprotectant is done in a fashion that is<br />
designed to reduce the risk <strong>of</strong> osmotic rupture and toxic effects on the cells <strong>of</strong> high osmolality and<br />
high concentrations <strong>of</strong> cryoprotectant.<br />
The choice <strong>of</strong> cryoprotectants <strong>use</strong>d depends on the cell development <strong>of</strong> the embryos. Propanediol<br />
is commonly <strong>use</strong>d for cleavage stage embryos and glycerol is commonly <strong>use</strong>d for the blastocyst<br />
stage <strong>of</strong> development. It is important to know that cell damage does not occur in the storage phase<br />
if stored in the liquid phase <strong>of</strong> liquid nitrogen. It is the freezing and thawing process that is crucial to<br />
cell survival.<br />
The embryos are placed into cryoprotectant solutions and then into containers such as straws or<br />
vials. A biological freezer then lowers the temperature around these containers at an accurately<br />
controlled rate. At the freezing point <strong>of</strong> the solution, ice crystal formation is induced <strong>by</strong> touching the<br />
containers with liquid nitrogen dipped forceps. This process is called seeding or ice nucleation. If<br />
this process is forgotten or inadequately performed the cells will suffer damage due to insufficient<br />
dehydration and intracellular ice formation. The formation <strong>of</strong> extra-cellular ice is a crucial event for the<br />
start <strong>of</strong> controlled water loss from the embryo.<br />
As the external concentration <strong>of</strong> the non-permeating solutes increases and the water freezes outside<br />
the embryo, water flows out, there<strong>by</strong> dehydrating the embryo. This process continues until the<br />
embryo reaches its freezing point. If the cooling is too fast the embryo will not become sufficiently<br />
dehydrated.<br />
If the embryo is thawed too slowly after freezing the super-cooled water will crystallise during the<br />
slow warming and intracellular ice will ca<strong>use</strong> damage. The rate <strong>of</strong> thaw is usually related to the cryoprotectant<br />
<strong>use</strong>d and the rate <strong>of</strong> cooling beyond –40°C. If the embryo freeze is a fast freeze, i.e. fast<br />
cooling rate to –40°C, the thaw rate will be fast i.e. plunging into +30°C water bath (500°C/min).<br />
Alternatively if the freeze is a slow freeze, i.e. slow cooling rate to –80°C the thaw must be performed<br />
in the freezing chamber at around +10°C /minute. Glycerol requires a rapid thaw.<br />
More information about the Cryo protocols can be found in:<br />
• Edgar DH, Karani J, Gook DA. Increasing dehydration <strong>of</strong> human cleavage-stage<br />
embryos prior to slow cooling significantly increases cryosurvival. Reprod Biomed<br />
Online. 2009 Oct;19(4):521-5<br />
• <strong>Gardner</strong> DK, Maybach J, Lane M (2001) Hyaluronan and RHSA increase blastocyst<br />
cryosurvival. Proc 17th World Congress on Fertility and Sterility, Melbourne. pp 226<br />
• Lane et al (2003) Cryo-survival and development <strong>of</strong> bovine blastocysts are enhanced <strong>by</strong><br />
culture with recombinant albumin and hyaluronan. Mol Reprod Dev 64:70–78<br />
• <strong>Gardner</strong> DK et al (2003) Changing the start temperature and cooling rate in a slowfreezing<br />
protocol increases human blastocyst viability. Fertil and Steril 79 (2): 407–410<br />
46<br />
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<strong>Vitrolife</strong> G-Series Manual 6.0
Cryopreservation <strong>of</strong> cleavage stage embryos using<br />
Freezekit MICRO A,B Cleave and Thawkit Cleave<br />
This<br />
A<br />
method <strong>of</strong> cryopreservation can be <strong>use</strong>d for pronuclear stage oocytes up to the 8-cell<br />
a)<br />
stage.<br />
f)<br />
b)<br />
a) b)<br />
Freezing procedure<br />
c) d)<br />
FreezeKit Cleave contains two solutions for freezing <strong>of</strong> pronuclear-stage oocytes and<br />
e)<br />
c)<br />
cleavage-stage embryos. The solutions consist <strong>of</strong> MOPS buffered solution and human<br />
d)<br />
serum albumin.<br />
B<br />
Equilibration solution, ES, contains no cryoprotectants<br />
b)<br />
a)<br />
Freezing solution, FS, contains 1,2 - propanediol and sucrose as cryoprotectants for<br />
dehydration <strong>of</strong> cells.<br />
d)<br />
c)<br />
Essentials<br />
b) b)<br />
• Only cryopreserve embryos <strong>of</strong> high quality since the thaw survival rate is related to<br />
initial quality <strong>of</strong> the embryo.<br />
• Check MICRO patient´s SETUP identity A,B and prepare all paperwork before you start the procedure.<br />
Hands<br />
A<br />
on<br />
B<br />
a)<br />
a)<br />
1 Label dishes and 1 straws, pipette appropriate volumes (0.5-1.0 ml) <strong>of</strong> ES and FS into<br />
respective 6 dishes. 2<br />
1<br />
4<br />
b)<br />
2<br />
5<br />
2 Equilibrate to room temperature. To avoid dilution, always transfer the embryos in a<br />
minimum 5 amount <strong>of</strong> solution. 3<br />
3<br />
6 b)<br />
4<br />
3 Place the embryos selected for freezing in ES and rinse properly. The embryos can stay<br />
in ES for 10 minutes.<br />
4 Transfer LOADSTRAW the embryos CRYO into the 1 FS and expose for 10 minutes. Loading <strong>of</strong> the prepared<br />
straws can start immediately after the embryos have been transferred to FS. The total<br />
exposure time for the embryos to the FS should be 10 minutes. Load the straws in<br />
accordance with manufacturer´s recommendation or as shown below.<br />
a) b) c) d) e) f) g) h) i)<br />
a) Syringe f) FS + embryos, 2–3 cm<br />
b)<br />
a)<br />
Plastic<br />
Syringe<br />
tube<br />
b) Plastic tube c) Cotton<br />
g) Air<br />
plug<br />
bubble<br />
sealing<br />
1/4 cm<br />
cement d) FS 2, 2cm e) Air bubble 1/4 cm<br />
f) FS 2 + embryos, 2–3 cm g) Air bubble 1/4 cm h) FS 2, 1 cm i) Seal: plastic plug<br />
c) Cotton plug sealing cement h) FS, 1 cm<br />
d) FS, 2cm i) Seal: plastic plug<br />
e) Air bubble 1/4 cm<br />
LOADSTRAW CRYO 2<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 Embryo Cryopreservation 47<br />
a) b) c) d) e) f) g) h) i)
5 Place the straws in the freezing machine at room temperature and initiate the freezing<br />
program.<br />
Use the following freezing program<br />
Starting temperature<br />
Step 1<br />
Step 2<br />
Step 3<br />
Step 4<br />
+18.0 to +25°C<br />
–2.0°C/min to –6.0°C<br />
Hold at –6.0°C and manually seed after 2 minutes. Keep the<br />
straw at –6.0°C for a total time <strong>of</strong> 10 minutes.<br />
Do not seed close to the embryo<br />
–0.3°C/min to –30°C<br />
–50°C/min to –150°C<br />
Plunge the straw into liquid nitrogen and arrange for storage at -196°C.<br />
Store submerged in LN 2 (not in the vapor phase)<br />
Thawing procedure<br />
ThawKit Cleave contains three solutions for thawing <strong>of</strong> pronuclear-stage oocytes and<br />
cleavage-stage embryos. The solutions consist <strong>of</strong> MOPS buffered solution and human<br />
serum albumin.<br />
Thawing solution 1, TS1, and Thawing solution 2, TS2, are buffered solutions containing<br />
decreasing concentrations <strong>of</strong> sucrose allowing removal <strong>of</strong> 1,2 -propanediol and gradual<br />
rehydration <strong>of</strong> the cells.<br />
Equilibration solution, ES, is a buffered solution that does not contain any cryoprotectants<br />
and is <strong>use</strong>d for final rehydration before culture.<br />
Essentials<br />
• Thaw straws one at a time.<br />
• Perform all steps at ambient temperature.<br />
• Check patient identity carefully and prepare all paperwork accordingly.<br />
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Hands on<br />
1 Label dishes, pipette appropriate volumes (0.5-1.0 ml) <strong>of</strong> TS1, TS2 and ES into<br />
respective dishes. Equilibrate to room temperature. To avoid dilution, always transfer<br />
embryos in a minimum amount <strong>of</strong> solution.<br />
2 Remove the straw from liquid nitrogen and expose to air for 30 seconds.<br />
3 Place the straw in a 30°C water bath for 45 seconds.<br />
4 Remove the straw and wipe it carefully. Open the straw using aseptic technique and<br />
gently expel the embryos. Expose the embryos to TS1 for 5 minutes.<br />
5 Move the embryos into TS2 and expose for 5 minutes.<br />
6 Move the embryos into ES and expose for 5 minutes.<br />
7 Transfer the embryos to accurately equilibrated culture medium, rinse embryos properly<br />
and culture according to standard laboratory procedure.<br />
Cryopreservation <strong>of</strong> cleavage stage embryos using<br />
Freezekit 1 and Thawkit 1<br />
This method <strong>of</strong> cryopreservation can be <strong>use</strong>d for oocytes, pronuclear stage oocytes up to<br />
8-cell embryos.<br />
The method is adapted from the original method <strong>of</strong> Testart et al (1986), using 1,2 propanediol<br />
(PrOH) as a permeating cryoprotectant. Sucrose is also <strong>use</strong>d as a non-permeating cryoprotectant.<br />
Sucrose is a large molecule that osmotically promotes dehydration during cooling and protects<br />
against cell lysis when thawing the embryos. A phosphate buffered solution is <strong>use</strong>d so that all steps<br />
may be performed outside the incubator and at ambient temperature.<br />
FREEZE-KIT1<br />
Cryo-PBS<br />
= Phosphate buffer solution with 25mg/mL Human serum albumin (HSA)<br />
Freeze solution 1 = FS1<br />
= 1.5M PrOH in Cryo-PBS<br />
Freeze solution 2 = FS2<br />
=1.5M PrOH + 0.1 M sucrose in Cryo-PBS<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 Embryo Cryopreservation 49
) b)<br />
MICRO A,B<br />
Example <strong>of</strong> freezing program for<br />
cleavage stage embryos<br />
A<br />
a)<br />
f)<br />
b)<br />
The duration <strong>of</strong> the freezing procedure including a) cryoprotectant equilibration b) is approximately<br />
2 h.<br />
Starting e) temperature<br />
d)<br />
Step 1<br />
B<br />
Step 2<br />
Step a) 3<br />
c) d)<br />
c) +18.0 to +25°C<br />
–2.0°C/min to –7.0°C<br />
HOLD AT –7.0°C for 10 min, SEED after 2 min<br />
b)<br />
–0.3°C/min to –30.0°C<br />
Step 4<br />
d) –30.0°C to below –80.0°C (at least 10°C/min)<br />
c)<br />
Remove straws and plunge into LN 2 , store submerged in LN 2 (not in the vapor phase).<br />
Essentials<br />
• Only cryopreserve embryos <strong>of</strong> high quality since the thaw survival rate is related to initial<br />
MICRO SETUP A,B<br />
quality <strong>of</strong> the embryo.<br />
• Check patient´s identity and prepare all paperwork before you start the procedure.<br />
A<br />
B<br />
a)<br />
a)<br />
1<br />
Hands on<br />
1<br />
6 2<br />
4<br />
1 Label dishes for freezing solutions, b)<br />
2<br />
5<br />
pipette appropriate volumes <strong>of</strong> Cryo-PBS, FS1, and<br />
FS2 into 5 respective dishes. 3 Equilibrate to ambient temperature.<br />
3<br />
6 b)<br />
4<br />
2 Observe and record detailed morphology. Rinse embryos for freezing in Cryo-PBS.<br />
3 Gently place embryos into FS1 for 10 minutes (never more than 20 minutes). The cells<br />
<strong>of</strong> the embryo will shrink and then re-equilibrate during this step.<br />
LOADSTRAW CRYO 1<br />
4 Move embryos across to FS2 and then load into straws <strong>by</strong> attaching the straw to a 1 mL<br />
syringe (syringes are connected to the straws <strong>by</strong> using 1 cm <strong>of</strong> silastic tubing). Remove<br />
the straws and seal, so that liquid nitrogen will not leak inside the straw.<br />
a) b) c) d) e) f) g) h) i)<br />
a) Syringe f) FS 2 + embryos, 2–3 cm<br />
b) a) Plastic Syringe tube b) Plastic tube c) Cotton g) plug Air bubble sealing 1/4 cement cm d) FS 2, 2cm e) Air bubble 1/4 cm<br />
c) f) Cotton FS 2 + embryos, plug sealing 2–3 cement cm g) Air bubble h) FS 1/4 2, cm 1 cm h) FS 2, 1 cm i) Seal: plastic plug<br />
d) FS 2, 2cm i) Seal: plastic plug<br />
e) Air bubble 1/4 cm<br />
LOADSTRAW CRYO 2<br />
50<br />
Embryo Cryopreservation<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
a) b) c) d) e) f) g) h) i)
5 Place into freezing chamber at ambient temperature and commence freezing program.<br />
6 Manually seed the straws at –7°C with liquid nitrogen (LN 2 ) cooled forceps close to the<br />
cotton plug.<br />
Do not seed the straw close to the embryos. Do not drop straw or shake it. If there are air<br />
bubbles in the straw it may reduce cell survival. Continue the freezing program.<br />
7 The freezing takes approximately 2 hours. Attention must be paid to the handling <strong>of</strong> the<br />
straws at low temperatures as they may break very quickly. Plunge into liquid nitrogen<br />
and arrange for storage at –196°C.<br />
Thawing program for cleavage stage embryos<br />
THAW-KIT 1<br />
Thaw solution 1 = TS1<br />
= 1.0 M PrOH + 0.2 M Sucrose in Cryo-PBS<br />
Thaw solution 2 = TS2<br />
= 0.5 M PrOH + 0.2 M Sucrose in Cryo-PBS<br />
Thaw solution 3 = TS3<br />
= 0.2 M Sucrose in Cryo-PBS<br />
Essentials<br />
• Thaw straws one at a time.<br />
• Perform all steps at ambient temperature.<br />
• Check patient identity carefully and prepare all paperwork accordingly.<br />
Hands on<br />
1 Identify patient and location <strong>of</strong> straw. Prepare all paperwork. Keep the straw under LN 2<br />
in small Dewar vessel until actual thawing. Label dishes for thaw solutions and pipette<br />
appropriate amounts <strong>of</strong> TS1, TS2, TS3 and Cryo-PBS.<br />
2 Remove straw and air thaw for 30 seconds. During this time handle the straws carefully<br />
and examine it for air bubbles, cracks in the seal and any leakage <strong>of</strong> LN 2 .<br />
3 Place in a +30°C water bath for 30 seconds. Remove and carefully wipe. Cut the plug<br />
end <strong>of</strong> the straw with sterile scissors, and attach it to a 1 mL syringe. Cut the other end<br />
carefully, do not shake straw or make air bubbles.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
Embryo Cryopreservation<br />
51
4 Gently expel the embryos into TS1. Observe the embryos coming out <strong>of</strong> the straw and<br />
if you do not see them all, quickly refill the straw and gently flush. Occasionally the<br />
embryos will stick to the sides <strong>of</strong> the straw.<br />
5 Incubate the embryos for 5 minutes in TS1.<br />
6 Gently move the embryos to TS2 for 5 minutes (approximately). Remember that these<br />
embryos are osmotically STRESSED and need to be handled VERY CAREFULLY.<br />
7 Move the embryos to TS3 for 5–10 minutes. The embryos will be stressed and the membranes<br />
will be very fragile.<br />
8 Place embryos in Cryo-PBS at ambient temperature for 6 minutes, followed <strong>by</strong> 4 minutes<br />
at 37°C on a heated stage. Do not place in the CO 2 incubator, as the buffer capacity<br />
<strong>of</strong> Cryo-PBS is insufficient for the 5 % CO 2 atmosphere.<br />
9 Embryos may be placed into equilibrated supplemented G-1 /G-1 PLUS (or<br />
G-2 /G-2 PLUS if the embryos were cryopreserved on day 3). Assess and record survival<br />
and morphology compared to pre-freeze. Embryos may be transferred immediately<br />
or left for further culture. Pronuclear oocytes should be cultured overnight in G-1 /G-1 <br />
PLUS and only transferred if normal cleavage has occurred.<br />
10 The thaw-cycle is very important to the success <strong>of</strong> embryo cryopreservation. Ensure that<br />
the embryo transfer is performed on the appropriate day. Embryos may be replaced in a<br />
natural or artificial cycle.<br />
Cryopreservation <strong>of</strong> blastocyst stage embryos<br />
G-FreezeKit Blast<br />
Supplement with either 50 mg/mL G-MM or 100 mg/mL HSA-solution :<br />
Blastocyst incubation medium (BIM)<br />
= 9.0 mL BIM + 1.0 mL G-MM or 1.0 mL HSA-solution .<br />
Blastocyst freezing solution 1 (BFS 1)<br />
= 9.0 mL BFS 1 + 1.0 mL G-MM or 1.0 mL HSA-solution .<br />
Blastocyst freezing solution 2 (BFS 2)<br />
= 9.0 mL BFS 2 + 1.0 mL G-MM or 1.0 mL HSA-solution .<br />
BIM is based upon a modified MOPS-buffered G-2 medium without sucrose and glycerol.<br />
BFS1 contains 100 mM <strong>of</strong> sucrose and 5 % glycerol<br />
BFS2 contains 200 mM <strong>of</strong> sucrose and 10 % glycerol<br />
52<br />
Embryo Cryopreservation<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
Example <strong>of</strong> freezing program for blastocyst stage<br />
embryos<br />
Starting temperature<br />
Step 1<br />
Step 2<br />
Step 3<br />
–6°C<br />
Seed after 2 minutes<br />
HOLD for 10 minutes<br />
Cool at 0.5°C/min to –32°C<br />
Remove straws and plunge into LN 2 , store submerged in LN 2 (not in the vapor).<br />
Essentials<br />
• Only cryopreserve blastocysts <strong>of</strong> high quality (≥ 3BB) since the thaw survival rate is<br />
related to the initial quality <strong>of</strong> the embryo.<br />
• Check patient identity and prepare all paperwork before the procedure is started.<br />
Hands on<br />
1 Label dishes for BIM, BFS 1 and BFS 2. Pipette 800 µL volumes <strong>of</strong> BIM, BFS 1 and<br />
BFS 2 into respective dishes. Equilibrate to +20 ± 5°C.<br />
2 Observe and record detailed morphology. Rinse blastocysts for freezing in BIM.<br />
3 Gently place embryos into BFS 1 for 10 minutes.<br />
4 Move embryos across to BFS 2 for 7 minutes.<br />
5 Use minimal volumes when moving embryos.<br />
6 During this 7 minutes rinse the freezing straw with BFS 2 in a separate dish/well.<br />
7 After 7 minutes, transfer the blastocysts to a new well with BFS 2 for loading into<br />
straws.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
Embryo Cryopreservation<br />
53
a) Syringe b) Plastic tube c) Cotton plug sealing cement d) FS 2, 2cm e) Air bubble 1/4 cm<br />
f) FS 2 + embryos, 2–3 cm g) Air bubble 1/4 cm h) FS 2, 1 cm i) Seal: plastic plug<br />
8 Load the blastocysts into pre-rinsed straws <strong>by</strong> attaching the straw to a 1 mL syringe<br />
(syringes can be connected to the straws <strong>by</strong> using 1 cm <strong>of</strong> silastic tubing). Remove the<br />
LOADSTRAW CRYO 2<br />
straws and seal, so that liquid nitrogen will not leak inside the straw.<br />
Do not exceed 15 minutes in BFS 2.<br />
a) b) c) d) e) f) g) h) i)<br />
a) Syringe f) BFS 2 + embryos, 2–3 cm<br />
b) a) Plastic Syringe tube b) Plastic tube c) Cotton g) plug Air bubble sealing 1/4 cement cm d) BFS 2, 2cm e) Air bubble 1/4 cm<br />
c) f) Cotton BFS 2 + plug embryos, sealing 2–3 cement cm g) Air bubble h) BFS 1/4 2, 1 cm cm h) BFS 2, 1 cm i) Seal: plastic plug<br />
d) BFS 2, 2cm i) Seal: plastic plug<br />
e) Air bubble 1/4 cm<br />
9 Immediately place into freezing chamber at –6°C.<br />
10 Wait 2 minutes.<br />
11 Manually seed the straws at –6°C with LN 2 cooled forceps at the top <strong>of</strong> the column<br />
containing the embryos. Do not seed the straw close to the embryos. Do not drop straw<br />
or shake it. If there are air bubbles in the straw, cell survival may be reduced. Hold the<br />
temperature for a further 10 minutes and then initiate the freezing program.<br />
12 The freezing takes approximately 50 minutes. Attention must be paid to the handling <strong>of</strong><br />
the straws at low temperatures as they may thaw very quickly. Plunge into liquid nitrogen<br />
and arrange for storage at –196°C.<br />
Thawing Program for blastocyst stage embryos<br />
G-ThawKit Blast<br />
Supplement with either 50 mg/mL G-MM or 100 mg/mL HSA-solution :<br />
Blastocyst thaw solution 1 (BTS 1)<br />
= 9.0 mL <strong>of</strong> BTS 1 + 1.0 mL G-MM or 1.0 mL HSA-solution <br />
Blastocyst thaw solution 2 (BTS 2)<br />
= 9.0 mL <strong>of</strong> BTS 2 + 1.0 mL G-MM or 1.0 mL HSA-solution <br />
Blastocyst thaw solution 3 (BTS 3)<br />
= 9.0 mL <strong>of</strong> BTS 3 + 1.0 mL G-MM or 1.0 mL HSA-solution <br />
Blastocyst incubation medium (BIM)<br />
= 9.0 mL <strong>of</strong> BIM + 1.0 mL G-MM or 1.0 mL HSA-solution <br />
54<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
BTS 1 contains 200 mM <strong>of</strong> sucrose and 10 % glycerol<br />
BTS 2 contains 100 mM <strong>of</strong> sucrose and 5 % glycerol<br />
BTS 3 contains 100 mM <strong>of</strong> sucrose.<br />
BIM is based upon a modified MOPS-buffered G-2 medium without sucrose and glycerol.<br />
Hands on<br />
All procedures take place at room temperature.<br />
1 Label dishes for BIM, BTS 1, BTS 2, and BTS 3. Pipette 800 µL volumes <strong>of</strong> BIM, BTS<br />
1, BTS 2 and BTS 3 into respective dishes or wells. Equilibrate to +20 ± 5°C.<br />
2 Fill cryo-dewer with LN 2 and quickly remove canes from cryo-storage tanks and place<br />
into LN 2 dewer.<br />
3 Remove straw from cane using forceps and thaw in air for 10 seconds.<br />
4 Place straw in a 30°C water bath for 30 seconds.<br />
5 Remove straw from water bath and dry thoroughly with a wipe. Cut the cotton end <strong>of</strong><br />
the straw and insert cut end into a 1 mL syringe. The syringe will be <strong>use</strong>d with the straw<br />
inserted into it to expel the embryos.<br />
6 Cut the other end <strong>of</strong> the straw and insert cut end into BTS 1. While looking through the<br />
microscope, expel embryos into BTS 1 using the syringe. Expel any additional liquid on<br />
to the dish cover and save to search through if all embryos are not recovered in BTS 1.<br />
The time taken from removing the straw from the waterbath to expelling into BTS 1<br />
should be minimal (less than 1 minute). Only one straw should be thawed at a time.<br />
7 Using a pulled pipette that is just slightly bigger than the embryos, move the embryos<br />
immediately into BTS 2 for 5 minutes. Transfer as little media as possible.<br />
8 Transfer embryos to BTS 3 for 5 minutes.<br />
9 Transfer embryos to BIM for 5 minutes.<br />
10 Transfer embryos to a second dish <strong>of</strong> BIM warmed on a stage warmer at +37°C for<br />
5‐minutes.<br />
11 Wash embryos in pre-equilibrated supplemented G-2 /G-2 PLUS once in the well <strong>of</strong> a<br />
1-well dish and place embryos into culture.<br />
12 Embryos should be transferred to the uterus in equilibrated EmbryoGlue ® or in equilibrated<br />
supplemented G-2 /G-2 PLUS.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 Quality Control Program 55
Quality<br />
Control Program<br />
All raw materials <strong>use</strong>d for manufacturing <strong>Vitrolife</strong> products are dedicated for human<br />
medical <strong>use</strong> and whenever applicable are <strong>of</strong> US Pharmacopeia (USP) and European<br />
Pharmacopoeia (Ph Eur) grade. Each lot <strong>of</strong> raw materials and final products are tested and<br />
evaluated <strong>by</strong> stringent quality control procedures. All production takes place in a manufacturing<br />
environment classified for aseptic production that is under strict control and monitoring.<br />
Quality Control, together with quality assured operations (ISO 13485:2003 and 21<br />
CFR Part 820:QSR) result in excellent LOT-to-LOT consistency.<br />
Physicochemical, biological and functional tests are performed according to the characteristics<br />
<strong>of</strong> each product. In addition to recognised standards for test performance, internal<br />
Standard Operating Procedures according to the Quality Handbook <strong>of</strong> <strong>Vitrolife</strong> are followed.<br />
The Quality Control Program <strong>of</strong> <strong>Vitrolife</strong> includes all necessary tests to guarantee<br />
both the safety and efficacy <strong>of</strong> the media.<br />
All instruments and standard solutions <strong>use</strong>d in the Quality Control system are acquired from<br />
qualified manufacturers.<br />
Validation and calibration<br />
All quality control procedures performed <strong>by</strong> <strong>Vitrolife</strong> are performed in accordance with<br />
quality assured operations (ISO 13485:2003 and 21 CFR Part 820:QSR) <strong>by</strong> highly<br />
qualified personnel. All methods and equipment <strong>use</strong>d are subject to a continuous and<br />
extensive validation and calibration program.<br />
56<br />
Quality Control Program<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
Physicochemical tests<br />
pH<br />
The pH measurement is performed using a validated method according to USP and Ph<br />
Eur. The range <strong>of</strong> acceptance for each product is kept as small as possible, considering the<br />
nature <strong>of</strong> the product and its intended <strong>use</strong>. Samples are pre-incubated at the appropriate<br />
temperature and atmosphere before measurement. Media equilibration at 6 % CO 2 is<br />
achieved <strong>by</strong> the <strong>use</strong> <strong>of</strong> a gas mixture with a certified CO 2 level.<br />
Osmolality<br />
The osmolality measurement is performed according to USP and Ph Eur using a validated<br />
method based on freezing-point depression. The range <strong>of</strong> acceptance for each product is<br />
kept as small as possible, considering the nature <strong>of</strong> the product and its intended <strong>use</strong>.<br />
Biological tests<br />
Bacterial endotoxin<br />
The absence <strong>of</strong> toxic levels <strong>of</strong> endotoxins is verified for all raw materials and each LOT<br />
manufactured. The validated bacterial endotoxin test (LAL assay) that is <strong>use</strong>d <strong>by</strong> <strong>Vitrolife</strong><br />
is the most sensitive <strong>of</strong> the currently <strong>use</strong>d methods for endotoxin testing with a minimum<br />
sensitivity <strong>of</strong> 0.005 EU or IU/mL. The validated procedure is performed in accordance with<br />
USP, Ph Eur, and FDA guideline “Guideline on validation <strong>of</strong> the limulus amebocyte lysate<br />
test as an end-product endotoxin test for human and animal parenteral drugs, biological<br />
products, and medical devices”.<br />
Sterility test - membrane filtration<br />
The sterility <strong>of</strong> all media is confirmed <strong>by</strong> a membrane filtration in accordance with USP and<br />
Ph Eur. The test period for media is 2 weeks and for oil 3 weeks. No detectable bacterial or<br />
fungal growth is accepted, neither in fluid thioglycollate medium nor in soya-bean casein<br />
digest medium. A strict requirement <strong>of</strong> the sterility assurance level (SAL) provides a safe<br />
application for <strong>use</strong>rs.<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 Quality Control Program 57
Functional tests<br />
Mo<strong>use</strong> embryo assay<br />
Mo<strong>use</strong> embryo assay (MEA) is a functional test method. All media, medium components<br />
and critical devices <strong>use</strong>d for media manufacturing are tested <strong>by</strong> culturing one-cell stage<br />
mo<strong>use</strong> embryos to the expanded blastocyst stage. The developmental stages <strong>of</strong> the<br />
embryos are recorded according to the specifics <strong>of</strong> each test. The safety and efficacy<br />
<strong>of</strong> the media is determined <strong>by</strong> observing the number <strong>of</strong> embryos that develop to defined<br />
developmental stages with appropriate cell numbers in a predetermined time period. In<br />
addition, the determination <strong>of</strong> cell numbers and the <strong>use</strong> <strong>of</strong> specific cut <strong>of</strong>f values set for all<br />
products increase the sensitivity <strong>of</strong> the MEA as cell numbers <strong>of</strong> the blastocysts are linked to<br />
their subsequent viability upon transfer to the uterus.<br />
Validation <strong>of</strong> Mo<strong>use</strong> Embryo Assay for Screening Medical Devices in Clinical<br />
ART<br />
A bioassay, such as the one cell Mo<strong>use</strong> Embryo Assay (MEA), is required for laboratory<br />
certification <strong>by</strong> the College <strong>of</strong> American Pathologists which governs Reproductive Clinics<br />
in the United States. All commercial media and those media made in ho<strong>use</strong>, must be evaluated<br />
prior to clinical <strong>use</strong> <strong>by</strong> such an assay. 1 The MEA is the most widely <strong>use</strong>d assay for<br />
media components, culture media, and equipment <strong>use</strong>d in clinical ART. 2 The MEA has been<br />
proven effective in screening for several potential embryo toxins present in the equipment<br />
<strong>use</strong>d in clinical work, new LOTs <strong>of</strong> oil <strong>use</strong>d as a media overlay, consumables and each new<br />
LOT <strong>of</strong> medium. Many steps have been taken to maximise the sensitivity <strong>of</strong> the MEA. For<br />
example, when testing contact materials we <strong>use</strong> media without albumin as it can chelate<br />
potential embryo-toxins. Furthermore, we <strong>use</strong> 1-cell mo<strong>use</strong> embryos as opposed to 2-cell<br />
stage. 3<br />
Determining cell numbers <strong>of</strong> blastocysts in the MEA is indicative <strong>of</strong> embryo viability.<br />
As shown in the figure below, fetal development is proportional to blastocyst cell number. 4<br />
Using the MEA it is possible to quantitate the product properties <strong>of</strong> culture media <strong>use</strong>d in<br />
clinical ART.<br />
REFERENCES<br />
1. College <strong>of</strong> American Pathologists (1998), Reproductive Laboratory-<br />
Section 90 Proposed Checklist, p26<br />
2. <strong>Gardner</strong> DK and Lane M (1999), Embryo culture systems. Handbook <strong>of</strong><br />
In Vitro Fertilisation (Second <strong>Edition</strong>), Eds. Trounson AO and <strong>Gardner</strong> DK,<br />
CRC Press, Boca Raton p 205-264.<br />
3. Davidson, A., Vermesh, M., Lobo, R.A., and Paulson, RJ (1998), Mo<strong>use</strong><br />
embryo culture as quality control for human in vitro fertilisation: the one-cell<br />
versus the two-cell model. Fertil. Steril., 49: 516-521.<br />
4. Lane M. and <strong>Gardner</strong> DK (1997), Differential regulation <strong>of</strong> mo<strong>use</strong> embryo<br />
development and viability <strong>by</strong> amino acids. Reprod Fertil., 109: 153-164.<br />
58<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
G-Series and related products from <strong>Vitrolife</strong><br />
Product 510(k) # Intended purpose<br />
G-MM K021894 G-MM contains Recombinant Human Albumin solution<br />
(50 mg/mL) and is intended for <strong>use</strong> in assisted<br />
reproductive procedures which include gamete and<br />
embryo manipulation. These procedures include the <strong>use</strong><br />
<strong>of</strong> G-MM as a supplement for culture medium.<br />
Not for <strong>use</strong> as an injectable product.<br />
HSA-solution K021896 HSA-solution contains Human Serum Albumin solution<br />
(100 mg/mL) and is intended for <strong>use</strong> in assisted<br />
reproductive procedures which include gamete and<br />
embryo manipulation. These procedures include the <strong>use</strong><br />
<strong>of</strong> HSA-solution as a supplement for culture medium.<br />
Not for <strong>use</strong> as an injectable product.<br />
CAUTION: All blood products should be treated as potentially<br />
infectious. Source material from which this product was derived was<br />
found negative when tested in accordance with current FDA required<br />
tests, HIV, types 1 and 2; HBV; HCV, and HTLV types I and II. No<br />
known test methods can <strong>of</strong>fer assurance that products derived from<br />
human blood will not transmit infectious agents.<br />
G-IVF K081116 Medium for preparation and handling <strong>of</strong> gametes<br />
G-IVF PLUS<br />
and for in vitro fertilisation.<br />
G-1 K081114 Medium for culture <strong>of</strong> embryos from the pronucleate<br />
G-1 PLUS stage to day 2 or day 3.<br />
G-2 K081117 Medium for culture <strong>of</strong> embryos from day 3<br />
G-2 PLUS<br />
to the blastocyst stage.<br />
EmbryoGlue® K031015 Medium for embryo transfer.<br />
G-MOPS K081115 Medium for handling and manipulating<br />
G-MOPS PLUS<br />
oocytes and embryos in ambient atmosphere.<br />
G-GAMETE K021893 For handling and manipulating oocytes<br />
and embryos in ambient atmosphere.<br />
G-RINSE K022295 Solution for rinsing <strong>of</strong> contact materials and for<br />
washing <strong>of</strong> the cervix. Not for culture.<br />
continued on next page<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 G-Series and related products from <strong>Vitrolife</strong> 59
Product 510(k) # Intended purposee<br />
G-FreezeKit Blast K032154<br />
Media for freezing <strong>of</strong> blastocyst stage embryos.<br />
G-ThawKit Blast K032155 Media for thawing <strong>of</strong> blastocyst stage embryos.<br />
G-PGD K033101 Medium for embryo biopsy.<br />
SpermGrad K023403 For gradient sperm separation.<br />
HYASE * K000627 For removal <strong>of</strong> cumulus cells.<br />
ICSI * K043116 For the immobilization and isolation <strong>of</strong> sperm prior to<br />
intracytoplasmic sperminjection, ICSI.<br />
FREEZEKIT 1 * K000623 For freezing <strong>of</strong> cleavage stage embryos.<br />
THAWKIT 1 * K000618 For thawing <strong>of</strong> cleavage stage embryos.<br />
FreezeKit Cleave * ✝<br />
Solutions for freezing <strong>of</strong> pronuclear oocytes and<br />
cleavage-stage embryos.<br />
ThawKit Cleave * ✝<br />
Solutions for thawing <strong>of</strong> frozen pronuclear oocytes and<br />
cleavage-stage embryos.<br />
OVOIL K991351 For covering <strong>of</strong> medium during in vitro fertilisation and<br />
micro-manipulation procedures.<br />
Additional products supplied <strong>by</strong> <strong>Vitrolife</strong><br />
IVF * K991348 For in vitro fertilisation, culture and transfer<br />
<strong>of</strong> embryos.<br />
ASP * K991345 For oocyte retrieval and rinsing (follicle flushing).<br />
SpermRinse * K000621 For sperm preparation.<br />
CCM * For For culture from day 3 to blastocyst stage and transfer.<br />
* Not available in Australia<br />
✝ Not available in the US<br />
research<br />
<strong>use</strong> only<br />
60<br />
G-Series and related products from <strong>Vitrolife</strong><br />
<strong>Vitrolife</strong> G-Series Manual 6.0
G-Series : Media Contents<br />
Medium Antibiotic MOPS NaHCO 3 Amino acids Vitamins Hyaluronan<br />
G-RINSE Gentamicin – Yes – – –<br />
G-MOPS / Gentamicin Yes Yes Non ess – –<br />
G-MOPS PLUS<br />
G-IVF / Gentamicin – Yes Non ess – –<br />
G-IVF PLUS<br />
G-GAMETE Gentamicin Yes Yes Non ess – –<br />
G-1 / G-1 PLUS Gentamicin – Yes Non ess – Yes<br />
G-2 / G-2 PLUS Gentamicin – Yes Non ess +Ess Yes Yes<br />
EmbryoGlue® Gentamicin – Yes Non ess +Ess Yes Yes<br />
G-FreezeKit Blast Gentamicin Yes Yes Non ess – –<br />
G-ThawKit Blast Gentamicin Yes Yes Non ess – –<br />
FreezeKit Cleave Gentamicin Yes Yes Non ess – Yes<br />
ThawKit Cleave Gentamicin Yes Yes Non ess – Yes<br />
G-PGD Gentamicin Yes Yes Non ess – –<br />
<strong>Vitrolife</strong> G-Series Manual 6.0 Media contents G-Series 61
Instruments <strong>by</strong> <strong>Vitrolife</strong><br />
For further information and ordering see www. vitrolife.com<br />
The availability <strong>of</strong> these Instruments may be subject to National Regulatory Requirements.<br />
Product 510(k) # Intended purpose<br />
Follicle Aspiration K991273 The Follicle Aspiration Set is a sterile, single <strong>use</strong> device<br />
Needles, single,<br />
which is intended for flushing and /or aspiration <strong>of</strong><br />
double and<br />
oocytes from the ovarian follicle<br />
luer lumen needles<br />
with several lengths<br />
and diameters<br />
Denudation K991700 The denudation pipettes are intended for removal <strong>of</strong> the<br />
Pipettes, several<br />
cumulus cell layers <strong>of</strong> the oocyte<br />
diameters<br />
Transfer Pipettes, K991700 The Transfer pipettes are intended for manipulation and<br />
two diameters<br />
transfer <strong>of</strong> oocytes, embryos and blastocysts, or to check<br />
fertilisation<br />
ICSI Pipettes K991700 The ICSI Pipettes are intended for intra cytoplasmic<br />
w/o spike,<br />
single sperm injection <strong>of</strong> oocytes<br />
Several diameters,<br />
angles and lengths<br />
ICSI Pipettes K991104 The ICSI Pipettes are intended for intra cytoplasmic<br />
w spike, Several<br />
single sperm injection <strong>of</strong> oocytes<br />
diameters,<br />
angles and lengths<br />
Holding Pipettes, K991700 The Holding pipettes are intended to hold the oocyte or<br />
Several diameters, K991104 embryo in position with the application <strong>of</strong> vacuum during<br />
angles and lengths<br />
intracytoplasmic single sperm injection or other micro<br />
manipulating procedures.<br />
Blastomere Biopsy K022643<br />
Pipette, several<br />
diameters and<br />
angles<br />
The Blastomere biopsy pipettes are intended to conduct<br />
a blastomere biopsy which may be done in order to<br />
perform pre-implantation genetic diagnosis <strong>of</strong> the genetic<br />
material in the biopsied cell(s).<br />
62 Instruments <strong>by</strong> <strong>Vitrolife</strong><br />
<strong>Vitrolife</strong> G-Series Manual 6.0
Product 510(k) # Intended purposee<br />
Partial Zona K991700 The Partial zona dissection pipettes are intended to make a<br />
Dissection<br />
hole in the zona pellucida to enable embryo assisted<br />
Pipette<br />
hatching.<br />
Hatching Pipette, K991700 The Assisted hatching pipettes are intended to make a<br />
two diameters<br />
hole in the zona pellucida to enable embryo assisted<br />
and lengths<br />
hatching.<br />
Microcell --- The Microcells are intended for counting <strong>of</strong> spermatozoa<br />
Sperm counting Not<br />
chamber<br />
Medical<br />
Device<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
Instruments <strong>by</strong> <strong>Vitrolife</strong><br />
63
Precautions and warnings<br />
Do not <strong>use</strong> if product appears cloudy.<br />
To preserve sterility, <strong>Vitrolife</strong> recommends that it be opened and <strong>use</strong>d only with aseptic<br />
technique.<br />
Media bottle should not be stored after opening. Discard excess media after completion <strong>of</strong><br />
procedure.<br />
For external <strong>use</strong> only. Not for <strong>use</strong> as an injectable product.<br />
Caution: US Federal law restricts this device to sale <strong>by</strong> or on the order <strong>of</strong> a physician.<br />
CAUTION: All blood products should be treated as potentially infectious. Source material<br />
from which this product was derived was found negative when tested in accordance with<br />
current FDA required tests, HIV, types 1 and 2; HBV and HCV. No known test methods can<br />
<strong>of</strong>fer assurance that products derived from human blood will not transmit infectious agents.<br />
The risks <strong>of</strong> reproductive toxicity and developmental toxicity for IVF media, including<br />
G5 Series <strong>of</strong> IVF Media, have not been determined and are uncertain.<br />
Further information<br />
This written material contains limited information regarding <strong>Vitrolife</strong> products. The<br />
information on various tests and clinical trials herein is only a summary provided for<br />
background information purpose about these products. This information should not be<br />
considered as complete, nor is it a substitute for the advice <strong>of</strong> a physician. Therefore, it is<br />
important to discuss the included information with a physician. If you are a physician, you<br />
are recommended to revert with <strong>Vitrolife</strong> or its authorised agents or distributors for complete<br />
information on products and procedures related herein.<br />
Moreover, this written material contains information relating in particular to health, suitability,<br />
the medical domain and various kinds <strong>of</strong> medical treatment reserved exclusively for <strong>use</strong> <strong>by</strong><br />
human beings.<br />
All products mentioned in this written material may not be available in all countries. For<br />
64 Precautions and warnings<br />
<strong>Vitrolife</strong> G-Series Manual 6.0
more information contact <strong>Vitrolife</strong> or the <strong>Vitrolife</strong> distributor. The recommendations for <strong>use</strong><br />
<strong>of</strong> the products may have changed since the printing <strong>of</strong> this written material. For the latest<br />
information please consult the <strong>Vitrolife</strong> homepage (www.vitrolife.com).<br />
Please note that all purchases <strong>of</strong> <strong>Vitrolife</strong> media and instruments products from <strong>Vitrolife</strong> are<br />
subject to the General Sales terms available on <strong>Vitrolife</strong>’s homepage (www.vitrolife.com)<br />
and in cases <strong>of</strong> conflicting contents, the General Sales Terms prevails.<br />
All IVF Media and Swemed IVF Instruments carry the European CE mark. With two<br />
exceptions (CCM and Stem Cell Cutting Tool) all products are also cleared for sale in<br />
the US.<br />
The information in this manual was correct at the time <strong>of</strong> printing.<br />
Latest updates can be found at www.vitrolife.com<br />
Correspondence<br />
<strong>Vitrolife</strong> welcomes any comments on the text in the Manual.<br />
If you have questions or need further explanations, please contact us:<br />
Europe, Middle East, Asia/Pacific<br />
Tel: +46-31-721 80 20 support line<br />
Fax: +46-31-721 80 99<br />
E-mail: support.fertility@vitrolife.com<br />
Address: <strong>Vitrolife</strong> Sweden AB<br />
Box 9080<br />
SE-400 92 Göteborg<br />
Sweden<br />
Internet<br />
http://www.vitrolife.com<br />
Americas<br />
Tel: 866-VITRO US (866-848-7687)<br />
Fax: 866-VITROFAX (866-848-7632)<br />
E-mail: support.us.fertility@vitrolife.<br />
com<br />
Address: <strong>Vitrolife</strong> Inc.<br />
3601 South Inca Street<br />
Englewood<br />
Colorado 80110<br />
USA<br />
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
Correspondence<br />
65
66 <strong>Vitrolife</strong> G-Series Manual 6.0
<strong>Vitrolife</strong> G-Series Manual 6.0<br />
67
Americas<br />
<strong>Vitrolife</strong> Inc., 3601 South Inca Street<br />
Englewood, Colorado 80110, USA<br />
Tel 866-848-7687 (866-VITRO US)<br />
Fax 866-848-7632 (866-VITROFAX)<br />
Europe, Middle East, Asia/Pacific<br />
<strong>Vitrolife</strong> Sweden AB, Box 9080<br />
SE-400 92 Göteborg, Sweden<br />
Tel +46-31-721 80 20, Fax +46-31-721 80 99<br />
E-support Americas<br />
support.us.fertility@vitrolife.com<br />
E-support Europe, Middle East, Asia/Pacific<br />
support.fertility@vitrolife.com<br />
E-mail<br />
fertility@vitrolife.com<br />
Internet<br />
http://www.vitrolife.com