1 8 . 0 5 6 . 2 0 0 3
Laboratory Manual
for Treatment and
Research in Reproduction.
This protocol is owned by Nordica and is only to be used in connection with instructions from
the Nordica embryologist.
Nordica takes no responsibility for any inflicted damages to things, gametes, embryos or persons
in treatment following this manual.
1
Embryo laboratories.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Preparation for oocyte pick-up
Oocyte pick-up
Insemination of the oocytes
Medium change
Embryo transfer
Embryo freezing
Thawing of embryos
Embryo destruction
Oocyte donation.
ICSI laboratories.
10. Utensils for the ICSI procedure, Preparation prior to ICSI
11. ICSI - procedure
12. TESE/ICSI
13. PESA/ICSI
14. Testis biopsy
Sperm laboratories.
15. Routine treatment of the sperm ejaculate
16. Preparation for the sperm preparations
17. Sperm Prep (Pure-sperm) preparation of the sperm
18. Swim - up
19. Sperm preparation for IUID
20. Sperm donor straw selection
21. Sperm - freezing procedures
Cleaning procedures.
22. Cleaning of the LAF bench
23. Disposal
24. Quality assurance
The following manual is only to be used with other instructions by the Nordica embryologist.
2
1. Preparation for oocyte pick-up.
1.1 Reagents: Flushing Medium 500 ml Cat. nr. 10830500.
Supplier: Medi Cult
Validity: Unopened, see expiry date on bottle.
Opened, 1 week. 100 ml. of sterile pipettes in the LAFbench, in 250ml.Falcon flasks.
Storage: 2-8C.
IVF-Medium 500 ml. Cat. nr. 10310500.
Supplier: Medi Cult
Validity: Unopened, see expiry date on bottle.
Opened, 1 week. 10, 20, 30 ml. of sterile pipettes in the LAFbench, in 50 ml. Falcon flasks.
Storage: 2-8C
1.2 Utensils: 20 ml. Braun Melsungen syringes Cat. nr. 4606736.
Supplier: Nomeco.
Nunc 4-chamber dishes Cat. nr. 00176 74 0A.
Supplier: Life Technologies.
Eppendorf multipipette and pipette tips Cat. nr. 0030-069-463.
Supplier: Radiometer.
Falcon medium culture flasks 50 ml. Cat. nr. 3014.
Falcon medium culture flasks 250 ml. Cat. nr. 3024.
Supplier: Becton Dickinson a/s
1.3
Preparation the day before aspiration.
The following items should be placed in the heating unit at 370C.
(PA means Per Aspiration)
Choose A or B procedure.
A. Test-tube procedure.
1 heating block for test-tubes
PA: 100 ml Flushing media or 100 ml Hepes Buffered IVF medium HAM F10 IVF medium with
L-Gluthamine (Life Technologies).
1 x needle
2o x test-tubes
B. Syringe procedure
1 heating block for syringes (placed in the hole between asp-room and lab)
PA: 7 x 20 ml. Braun syringes (no rubber) + 1 needle
60 ml Flushing media (Medicult) (Sucked up after aspiration to flush the needle.)
1 open medium container 150 ml.
1. Syringe 20 ml piston of plastic, Luer Lock
2. Speckles
3. Sterile water for washing
3
4. Sterile napkins
5. Claw forceps to help
6. Needle guide One per patient.
1.4
Place in the incubator:
(Each shelf in the incubator has a number marked on the outer door).
1. 3 x 4- wells Nunc dishes (if more than 12 eggs prepare 4) with the patient's name, civil nr.,
shelf nr. and dish nr. written - thus the patient chart and labels etc. have this code.
2. The Nunc 4 - well dishes are prepared with 500l IVF medium in each well and
3 x 500 l IVF medium in the central chamber. (with pipette placed in the middle)
3. The Nunc dishes are placed in the culture incubator for the next day. Only two patients on
each shelf.
(To be placed in the Nurse procedure)
Nurse - In the aspiration room - The day for oocyte pick ups - (Immediately before the
procedure).
7. The Flushing medium 30 ml is pourred out into the open medium container (heating unit).
8. The cover around the syringes is opened (keeping the items sterile) (heating unit).
9. Sterile gloves are taken on the hands.
4
2. Oocyte pick - up.
2.1 Reagents.
2.1.1 IVF-Medium 500 ml. Cat. nr. 10310500.
Supplier: Medi Cult
Validity: Unopened, see expiry date on bottle.
Opened, 1 week. 10, 20, 30 ml. of sterile pipettes in the LAF-bench, in 50 ml.
Falcon flasks.
Storage: 2-8C.
30 ml flush medium is used per asp.
2.2 Utensils:
2.2.1: 20 ml. Braun Melsungen syringes Cat. nr. 4606736.
Supplier: Nomeco.
2.2.2: Falcon Petri-dish 60 x15 mm. Cat. nr. 1007.
Supplier: Becton Dickinson a/s.
2.2.3: Nunc 4-chamber dishes Cat. nr. 00176 74 0A.
Supplier: Life Technologies.
2.2.4 Eppendorf pipettes and pipette tips 10-100 l Cat. nr. 0030-001-303.
2.2.5 Eppendorf multipipettes and pipette tips 0,1-2,0 ml. Cat. nr. 0030-001-311.
Supplier: Radiometer.
Heating block
Wild Heerbrugg stereo microscope
Wilovert microscope
Aspiration.
The Journal for the patient (chart)
2.0. Secretary produces proper patient file (Lab—file, +++)
2.1. Nurse Lab-file filled out with patient data and dates. When the patient is scheduled for
oocyte pick-up (2-3 days before), the lab file is placed in the rack for aspiration in the
laboratories. Note the estim nr. of oocytes.
2.2. Lab: The day before aspiration will the name and civil nr., as well as the estimated nr. of
oocytes retrieved be noted in the lab file by the embryologist together with the clinician.
Following the oocyte pick-up procedure, the number of the oocytes and the maturation of the
oocyte-cumulus complexes are noted in the chart.
The results of the sperm samples are also noted in the chart.
Patient identification procedure.
1. By arrival, semen sample is delivered to the lab.
2. When the clinician has cleared for oocyte pick-up, the patient is identified in the operating
room by the embryologist and the doctor.
3. The follicular fluid in the test-tubes are placed in the heating blocks, and is collected by the
embryologist.
4 The oocyte cumulus complexes are identified using the stereo microscope and Petri dishes
where the follicular fluid is placed in.
5. The oocytes are, after washing, placed in the Nunc 4 - well dishes and placed under the CO2
external incubator hood in the LAF and finally placed in the incubator.
6. The lid must be lifted on edge for 10 minutes to avoid condense.
7. The embryologist informs the clinician about the number of oocytes collected and notes this
on the lab file.
3. Insemination of the oocytes.
5
1. Approx. two hours after oocyte pick-up, will the oocytes be inseminated with purified and
cleaned motile spermatozoa (see procedure 17).
2. Check the ID Name and nr. of the sperm suspension.
3. The sperm suspension is adjusted to 2 mill. spermatozoa/ml i.e. 100.000 spermatozoa per
oocyte.
10. 4. The 4-well dishes with the oocytes are taken into the LAF bench and if all identification
marks on the chart, sperm suspension and on the dishes are identical, (checked by two
persons), the sperm suspension will be added i.e. 50 yl coresponding to 100.000
spermatozyoa/well.
5. Approx 2 hours later the oocytes are moved to a clean well in the same dish.
4. Medium changes.
Preparation for medium changes.
1. At the day for oocyte aspiration all dishes to be used for change the next day, shall be
prepared. All dishes are marked with patient identifications and kept with the specific patient
colour.
2. The dishes are filled with 500l IVF medium per well and 2 x 500l medium in the central
well.
3. The dishes are placed in the CO2 incubator until the next day.
Medium change.
24 hours after aspiration
1. The dishes with the fertilised oocytes + dishes prepared for medium changes are taken out of
the incubator into the LAF bench.
2. In the LAF bench, under the stereo microscope, all eggs will be transferred to the new dishes.
All dishes are kept under CO2 during this procedure as long as possible.
3. When all eggs have been transferred to the fresh medium - all embryos will be checked for
pronuclei formation. This information is noted for each single oocyte in the chart.
4. All dishes with embryos are again placed in the CO2 incubator with the lid lifted to ensure
equilibration with the gas in the incubator. After 10 minutes the lids are again placed in proper
position.
6
5. The embryo transfer procedure.
Utensils:
Wallace IVF-catheter Cat. nr. 1816.
Supplier: Helle Henriksen.
1 ml.Omnifix syringes Cat. nr. 916140-6.
Supplier: Nomeco
Transfer.
1. At the day of transfer the embryos will be assessed for transfer, freezing and destruction by the
embryologist and the doctor in the morning. The decisions and the cleavage stage and embryo
score are noted in the lab file.
2. Identification procedure – name and civil code
3. The patient is informed by the nurse, embryologist and doctor about the status of the embryos.
4. The embryologist loads the catheters according to the following rules:
The catheter is first flushed with IVF medium (well equilibrated). Again a second flush is carried
out, however only ½ of the volume in the syringe is expelled. First, when the doctor announces
that he is ready to do the transfer, the syringe will be emptied and the embryos loaded. The
embryos are placed as close as possible to the tip (0,5 - 1 cm) in a total of 10 to max 20l of
medium. Eventually marked with 2 small air drops.
The catheter is delivered to the doctor.
5. After the transfer, the embryologist receives the catheter to be checked for possible remaining
embryos by flushing the catheter in the 4-well dish.
When any difficulties arises during transfer, the embryologist return the embryos to the culture
dish and places it in the incubator until the clinician is ready again. Then, a new catheter is loaded
with the embryos.
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6. Embryo freezing.
Reagents:
Embryo Freezing Medium 4x10 ml.
F1 Embryo Freezing Medium
Cat. nr. 10790010.
F2 E.F.M med 1,5 M 1,2 Propanediol
Cat. nr. 10240010.
F3 E.F.M med 1,5 M 1,2 Propanediol and 0,1 M Sucrose
Cat. nr. 10250010.
Supplier: Medi Cult
Validity: Unopened, see expiry date on bottle.
Opened, 1 week. 1 ml. of sterile pipettes in the LAF-bench, in
6 ml. Falcon glass.
Storage: 2-8C
By opening the media F1, F2 and F3, distribute the 10 ml. to 10 tubes with 1 ml.
each. (Falcon 6 ml).
Utensils:
Nunc 4-chamber dishes Cat. nr. 00176 74 0A.
Supplier: Life Technologies.
1 ml. Omnifix syringes Cat. nr. 916140-6.
Supplier: Nomeco.
Eppendorf pipettes and pipette tips10-100 l Cat. nr. 0030-001-303.
Eppendorf multipipettes and pipette tips 0,1-2,0 ml Cat. nr. 0030-001311.
Supplier: Radiometer
Inner tubes 9,2 mm Cat. nr. see catalogue.
Supplier: Danske Kvægavleres Fællesindkøb
Propper Cat. nr. seecatalogue.
Supplier: Danish Cow-breading Center.
Freezing straws Cat. nr. ZA 483.
Supplier: Danish Cow-breading Center.
Freezer.
Wild Heerbrugg stereo microscope.
Freezing-tank
Forceps.
Stopwatch.
Wallace IUI-catheter Cat. nr. AIC 18.
Supplier: Helle Henriksen
(used for aspiration of the embryos into the straws) (Re-use)
Falcon glass 6 ml. Cat. nr. 2058.
Supplier: Becton Dickinson a/s
8
Embryo freezing procedure.
Straw, sealing and holders should be marked with the patient's identifications and date for
freezing. The colour and place is chosen according to the space in the freezing jar. If more than
one straw, the straws are numbered 1,2,3, etc.
These procedures have to be checked by two persons.
In connection with the transfer procedure, eventual eggs will then be selected for freezing.
The freezing media is applied to the 4-well chambers as follows:
well no. 1: F1
well no. 2: F2
well no. 3 and 4: F3
Approximately 500l per well.
The embryos to be frozen are washed in F1 and transferred to F2.
In F2 the are kept for 10-15 minutes and hereafter transferred to F3. After 10-15 minutes in F3
will the eggs and medium be loaded in the straws. A maximum of 3 eggs per straw.
F3
Air
F3 + embryos
air
F3
The sealing is applied and the prepared straw is placed in the freeze control system at 200C. After
18 minutes, you will be able to perform the seeding procedure at -6 oC. The freezing procedure
will take 1,5 hours.
The journal.
In the journal all data will be noted, including a freezing note.
The freezing records.
The patient will be recorded in the freezing journal and record.
The patient's ID including place in the freezing container with name number and colour. All this
information, should be checked by two persons.
The couple (man and woman), have to sign the freezing agreement before the actual freezing .
7. Embryo - thawing
Reagents:
Embryo Freezing Medium 4x10 ml
T1 E.F.M med 1,0 M 1,2 Propanediol og 0,2 M Sucrose
Cat. nr. 10410010
T2 E.F.M med 0,5 M 1,2 Propanediol og 0,2 M Sucrose
Cat. nr. 10420010
9
T3 E.F.M med 0,2 M Sucrose
Cat. nr. 10430010
Supplier: Medi Cult
Validity: Unopened, see expiry date on bottle.
Opened, 1 week. By opening the media T1, T2 and T3,
distribute the 10 ml. into 10 tubes with 1 ml. each. (Falcon 6
ml). sterile in the LAF-bench.
Storage: 2-8C
IVF-Medium 500 ml Cat. nr. 10310500
Supplier: Medi Cult
Validity: Unopened, see expiry date on bottle.
Opened, 1 week. 10, 20, 30 ml.of sterile pipettes in the LAFbench, in 50 ml. Falcon flasks.
Storage: 2-8C.
IVF-Medium without Pen/Strep. 60 ml. Cat. nr. 10950060.
Supplier: Medi Cult.
Validity: Unopened, see expiry date on bottle.
Opened, 1 week. 5, 10 ml. of sterile pipettes in the LAF-bench, in 50ml.
Falcon flasks.
Storage: 2-8C.
Utensils:
Nunc 4-chamber dishes Cat. nr. 00176 74 0A.
Supplier: Life Technologies.
1 ml. Omnifix syringes Cat. nr. 916140-6.
Supplier: Nomeco.
Eppendorf pipette and pipette tips 10-100 l Cat. nr. 0030-001-303.
Eppendorf multipipette and pipette tips 0,1-2,0 ml Cat. nr. 0030-001311.
Supplier: Radiometer
Wallace IUI-catheter Cat. nr. AIC 18.
Wallace IVF-catheter Cat. nr. 1816.
Supplier: Helle Henriksen.
Falcon glass 6 ml. Cat. nr. 2058.
Supplier: Becton Dickinson a/s
Biogel Gloves.
Supplier: C.I.K.
10
The day before thawing embryos.
1. The freezing journal and records are placed in the laboratories and 4-well dishes are marked
with the colour and patient's ID.
2. The wells are filled with the IVF medium.
The day of thawing.
1. The media T1, T2, T3, IVF medium is placed in a 4-chamber well marked with
patient name and ID nr. (LAF Bench)
2. At the freezing chart, it is confirmed which and where the straw to be thawed is placed. (see
lab freezing file)
The thawing procedure.
1.
2.
3.
4.
5.
6.
7.
8.
9.
The straws are identified in the freezing container and checked by two persons.
The straw is transported to the LAF-bench (max 45-sec) by hand.
Place the straw on the hot plate for 30 sec.
The sealing is cut off and the syringe (1ml) is applied. The flag at the other end is cut off.
The straw is then emptied in chamber 1 (T1 of the 4-well).
After 5-10 minutes the embryos are transferred to T2.
5-10 minutes later again transferred to T3 (well 3).
5-10 minutes later again transferred to T4 (well 4).
Hereafter, the embryos are transferred to the well-equilibrated 4-well Nunc dish with IVF
medium, made the day before.
10. The eggs are scored and left overnight in the incubator to cleave.
11. The straws and eggs are all checked by two persons.
12. The next morning, the eggs will again be scored.
All these information should be available for the chart and journal.
The lab-file.
In the journal the following information should be noted.
1. The next day : the state and score of the eggs after thawing.
2. Before transfer : the state and score of the eggs
3. Also how many eggs left in the freezer.
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8. Embryo destruction.
1. The patients’ eggs will, according to Danish law, be destroyed after 2 years.
2. The embryos are identified, by means of the freezing journal. The day for destruction is
noted in the lab file.
3. In the lab file and chart, the date for destruction is confirmed and the names of the two
embryologists doing this are also noted.
9. Oocyte donation.
The patient who wants to donate oocytes to another woman has to fulfil the following criteria:


The age of the donor shall be less than 36 years.
Shall have a minimum of 10 oocytes of her own after donation.
- From the nurse or clinician, the laboratory will be notified with name and civil number of the
recipient.
- The ova will be transferred as usually. The dishes marked, as described before, for aspiration
day.
- The insemination of the donated eggs takes place after 2 hours following the oocyte pick-up
procedure.
- The journals and charts are kept as normal.
On the donator's chart and journal, the number of oocytes donated is marked.
The donator should, at the day of donation (oocyte pick-up),
have drawn blood samples for HIV, Hepatitis.
No embryos may be transferred to the recipient before the negative answer from these
blood test are available.
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10. ICSI
Utensils:
Hyaluronidase is bought at Medi-Cult.
We have a standing order for 1 year, providing the laboratories with 2 x 10 ml. every Thursday.
The 2 x 10 ml are immediately placed in Falcon 10 ml. test-tubes, 1 ml. Hyaluronidase in each.
PVP and oil is bought at Medicult.
We have a standing order for 1 year, providing the laboratories with 2 ICSI - 100 ( 20 x 100
PVP) and 10 ovoil - 150 (10 x 150 ml. oil).
The pipettes are bought at SWE-MED LAB and Cook, and we are using the following:
Holding pipette
0.018 - 0.025
ICSI pipette
0.0048 - 0.0056
Denuding pipette
0,134 – 0145 (Sweamed)
NOTE: Remember to place an order in advance, the delivery time is very long.
Petri dishes.
The only ones to be used are the FALCON, cat. Nr. 1006.
Microscope.
Oil for the holding and ICSI pipette (tubing’s).
Sigma embryo tested cat. No. M - 8410.
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11. The ICSI procedure
11.1. Preparation the day before the ICSI procedure
 Oil.
Oil is incubated in the CO2 incubator in a 50 ml. Falcon bottle.
The amount of oil used is 25 ml. per patient for the following day.
 IVF and harvest medium. Medicult Flushing Medium (Hepes)
150 ml. buffered harvest medium per patient.
IVF medium for all patients.

Hyaluronidase.
1. Dish 1. Preparation of denudation - dishes. Hyaluronic acid in well nr. 1 and Medicult Sperm
Prep Medium (Hepes) medium in all other wells (500 l per well). The dish is placed in the
CO2 incubator until usage.
2. Dish 2. The injection dishes are then prepared. A maximum of 8 droplets placed in a circle of
Sperm Prep medium in a volume of 5-10 l. Each is placed in a circle in the Petri dish
(Falcon dish 1006). In the centre, a 10 l Sperm Prep droplet is placed. The droplets are
overlaid with 4 ml. oil and the dish is placed in the CO2 incubator again.
3. Dish 3. The culture dish is prepared with droplets 10yl IVF media in rows. To make it easy to
follow, the eggs are separated and covered with 4 ml. oil.
4. 4-well dish for aspiration - see preparation for oocyte pick-up pg.3.
11.2: Preparation day.
1. The cumulus oocyte complexes are kept in 4 well Nunc dishes with IVF medium from
aspiration until denudation (approx. 1 hour).
2. The microscope is prepared and made ready for the ICSI procedure itself. Injection and
holding pipettes are mounted and adjusted
3. The eggs are denuded following these instructions: 4 - 8 oocytes are placed in the Hyaluronic
acid ( well 1) and pipetted up and down using a pipette with a 50 l volume. This procedure
is stopped when most of the cumulus cells have detached from the ova. Then, the eggs are
transferred to the next well with hepes buffered IVF medium. The ova should not be in the
Hyaluronic acid solution for more than 1 minute. In the hepes buffered IVF medium the
remaining cumulus cells are removed by using a denudation pipette ( SWE-MED 0,134 0,145) The stripped oocytes are then transferred to well nr. 3. When all oocytes are cleaned
from cumulus cells, all eggs are then transferred to the ICSI dish 2. Max. 8 eggs per dish.
(Petri dish Falcon 1006, as mentioned above).
4. ICSI-dish 2. The center drop medium is removed and 10yl yl PVP is placed instead. Ad 2 yl
PVP of semen suspension to this drop.
11.3: The ICSI procedure
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1. The spermatozoa to be injected is captured. Catch the spermatozoa in the peripheral area of
the droplet. The spermatozoa are immobilised by rolling and touching the sperm-tail with the
pipette. It is important that the spermatozoa are completely immobilised before injection.
The immobilised sperm is sucked up into the pipette lumen with the tail first. The Petri dish
is then moved to the first egg for injection. The holding pipette is then lowered into the
droplet and the egg is fixed with the polar body at the 12 or 6 o’clock position.
2. The injection pipette is adjusted, thus allowing the penetration to take place in the centre of
the oocyte. This adjustment is controlled by gently touching the egg with the injection
pipette. If the oocyte roles around, this will mean that the pipette has to be adjusted. When
the injection pipette is positioned correctly, the focus is adjusted to be able to see the sperm
in the pipette. The sperm is then placed in the very tip end of the pipette. The pipette is then
forced into the oocyte. When well within the oocyte, a gentle suction is applied to ensure the
break of the oolemma. This is done by, visualising a tiny amount of the cytoplasm entering
the pipette. Immediately hereafter, the sperm is expelled into the oocyte and the pipette
retracted out of the oocyte. All eggs in the Petri dish are injected by the same method.
3. When all the eggs in the Petri dish have been injected, they are then transferred back into
Petri dish 3 and placed in the CO2 incubator.
4. The rest of the embryo culture system is identical to normal IVF procedures.
15
12. TESE and ICSI (Testicular/epididymal sperm extraction).
The day before the procedure, every preparation is mentioned for routine ICSI procedures, + 2
extra 15 ml. Falcon tubes with Sperm Prep (placed in the incubator).
The day for sperm aspiration and oocyte pick - up.
1. At the same time as the doctor and the nurse prepare for sperm retrieval, the embryologist
prepares the following in the laboratories:
A: 2. Falcon test-tubes with the patient's identifications are prepared with 2 droplets of Heparin
(see later).
B: 2 large Petri dishes containing 5 ml. of hepes buffered Sperm Prep with 4 - 5 droplets of
Heparin. These 2 Petri dishes are transported into the aspiration room and used for washing of
the testicular tissue and hereafter, placed in the other to be transported back to the laboratories.
2. In the lab with the tissue.
Wash the tissue and remove to a new dish with Sperm Prep to avoid blood cells (erytrocytes).
A: By means of the coverslides or 2 bend needles, the tissue is disintegrated by gentle traction
against the Petri dish bottom. When the tissue is disintegrated the dish is carefully checked using
the inverted ICSI microscope. As soon as spermatozoa are identified the clinician is notified, thus
he can stop the operative procedures. The head of the individual spermatozoa might be attached
to the Sertoli Cells, but gentle traction with the pipette will often release these sperms.
B: The medium from the Petri dish is taken over into a Flacon test-tube with the patient's ID. In
the sperm laboratories is the sample centrifuged and the pellet suspended again in 2 droplets of
medium.
Now the ICSI procedure can start:
1.
2.
3.
If there are very few spermatozoa you may find them and place them in the PVP drop on
a row before you transfer the eggs, in order to keep the eggs in the incubator for as long
as possible.
Just before the ICSI procedure, the oocytes are transferred to the injection dish. Only a
maximum nr. of 4 oocytes are transferred in these cases to every injection dish. In the rest
of the droplets (4 - 5 droplets) 1 - 2l sperm suspension is added.
The embryologist is now allowed to search for motile spermatozoa. When one is found it
is transferred with the injection pipette into the PVP droplets. Here, the sperm is cleaned
from debris and blood cells and further immobilised. Hereafter, the sperm is again taken
up into the injection pipette and the ICSI can be performed. The procedure is repeated
until all eggs are injected.
The rest of the procedure is as described for routine ICSI procedures.
16
13. PESA and ICSI (Percutan epididymal sperm aspirations).
Ask Svend – The procedure has been scrapped..
The preparation for this procedure is the same as for ICSI, with some modifications.
1. 1 Falcon test-tube, with the patien'ts ID and 2 droplets of Heparin is prepared. This test-tube
is going to be used for sperm cells.
2. 5 x 1 ml. syringes is loaded with sterile Sperm Prep - medium. These are delivered to the nurse
in the aspiration room.
3. The clinician has punctured the epididymidis with a “butterfly” G 23. The aspirate including
some of the IVF medium is expelled into the Falcon test tube. In the laboratories 20l of this
suspension is examined under the microscope for the presence of spermatozoa. This
microscopic procedure is done by 200 x magnification. If sperm is present the rest of the
procedure is as mention in the ICSI procedure. If not the procedure in the aspiration room is
repeated or the clinician prefers to do the TESE procedure.
The rest of the procedure as already described.
Utilities for PESA/TESA/MESA procedures.
Heparin: It is important to use Heparin form Leo Løven, Denmark 5000 i.u.per ml.
Formaldehyde for testis biopsy material.
17
14. Testis Biopsy.
The testis biopsy is received from the clinician in Sperm Prep (as for the TESA procedure). The
biopsy is rinsed in this Sperm Prep medium and transferred to another Sperm Prep medium
when blood has been removed. Hereafter, the biopsy is divided into two pieces. One to used in
the IVF laboratories and one for the pathology department.
The biopsy for the laboratories is disintegrated as described for TESA procedures and the
embryologist is looking for living spermatozoa. The results of this finding is noted in the
patient,s journal.
The biopsy for pathology is placed in a container with buffered formaldehyde and sent.
(Cautions: buffered formaldehyde may never be in the IVF lab).
18
15. Routine treatment of the sperm sample in the sperm
laboratories.
The sperm sample is received by an embryologist from the husband, with the name, ID etc. Both
the husband and the wife in treatment are double checked. The chart is also checked for correct
ID.
For every patient, the following is prepared in the laboratories:
- 1 test-tube for Pure Sperm or swim-up
- 1 test-tube for wash of the sperm sample after swim - up or Pure Sperm
- 1 test-tube for dilution
All these tubes are marked with the patient,s colour code and ID and checked by two persons.
NB: The supernatants form each sperm sample may first be destroyed after the insemination.
16. The day before the Sperm sample is collected.
The day before sperm collection the following items are prepared and made ready in the CO2
incubator:
For IVF / ICSI patients:
- 1 test-tube with Sperm Prep medium. (10 ml)
- 1 test-tube with Pure Spem 55%
- 1 test-tube with Pure Spem 80%
For IUIH / IUID patients:
- 1 test-tube with hepes buffered Sperm Prep medium. (10ml)
- 1 test-tube with Pure Spem 55%
- 1 test-tube with Pure Spem 80%
19
17. Preparation of the sperm sample.
The day before the Pure Sperm preparation of the sperm sample the following are placed in the
CO2 incubator:
- Test-tube with 55% Pure Sperm
- Test-tube with 80% Pure Sperm
- Test-tube with Pure Sperm medium (if the preparation is going to be used for IVF)
or, test-tubes with buffered Sperm Prep medium (if used for IUI).
(Remember not to tighten the lid.)
The Pure Sperm gradient is made in a test-tube (Per patient) with the patient's colour/ID etc. in
the following manner:
1-2 ml. of 55% Pure Sperm in the test-tube is carefully underlayered by 1-2 ml. of 80% Pure
Sperm, thus a clear meniscus can be identified.
A maximum of 4 ml. sperm ejaculate is placed on top of the Pure Sperm gradient. If more than 3
ml. of ejaculate is present, more then one gradient is used.
The gradient with the sperm is centrifuged at1500 rpm for 25 min.
The supernatant is then removed.
The sediment is suspended again in 2 ml. of IVF or buffered IVF medium and centrifuged at
1400 rpm for 5 - 10 minutes. This washing procedure is repeated once.
Following the washing procedure, the sediment will be suspended again in 1-2 ml Sperm Prep
medium. The sample is counted in a Makler chamber.
The Sperm suspension is adjusted to 2 mill. spermatozoa per/ml.
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18. Procedure for Swim - up for IVF or IUI.
(This procedure is replaced by 17. Pure Sperm)
The day before the sperm preparation, a test-tube with 10 ml. of Sperm Prep medium or hepes
buffered medium is placed in the CO2 incubator.
The preparation of the sperm sample must be initiated before 1 hour after ejaculation.
One or more test-tubes (depending on the sperm conc. and volume of the ejaculate) is marked
with the patient's ID.
In each test-tube, 2 ml. of Sperm Prep medium is placed and then, 2 ml. of raw sperm are
carefully underlayred.
The test-tubes are gassed and placed at 37 0C. for 1 hour in the CO2 incubator if the samples are
going to be used for IVF, or just in the warm bench if used for IUI.
Another empty test-tube is marked with the patient's ID. In this test-tube, the top layer of the
swim - up procedure is carefully placed.
This swim - up layer is centrifuged at 1500 rpm for 5 minutes and the supernatant is removed.
The sediment is suspended again in 1 ml. of medium. 1 droplet is placed in the Makler chamber.
The count is noted in the patient's chart.
19. Sperm preparation for IUID.
For IUID, Pure Sperm preparations are always used.
The straw to be chosen has to match the race, height, weight, eye colour and hair of the husband
for the couple to be treated.
One straw per patient. If the straw exhibits less than 12 mill./ml motile spermatozoa ,an extra
straw is taken. We only accept over 2 mill. motile spermatozoa after final preparation for IUID.
If the patient has to be inseminated for two consecutive days, the same donor is used for both
days.
The donor straw is removed from the freezing tank as fast as possible to avoid disturbance of the
remaining straws in the freezer.
The straw is cut at one end and left in a test-tube to thaw, for 15 minutes.
A droplet of the straw content is controlled in the Makler chamber. From here on, the procedure
is as for Pure Sperm preparations.
All ID and sperm counts are noted in the patient's chart as well as the ID for the donor used.
This has to checked by two persons.
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20. Procedures for selection of donor straw with sperm.
1. The straw to be chosen has to match the race, height, weight, eye colour and hair of the
husband for the couple to be treated.
2. If no straw matches the characteristics for the husband, new straws from the donor bank are
ordered.
3. When straws are ordered, ask for more than 5 different donors per request, to enable better
selection in the clinic.
4. When straws are received from the sperm bank, every straw and characteristics including
straw and donor number are noted in the freezing protocol and then placed in the freezing
storage tank. Here, the place and colour of the straw is also noted in the protocol. Everything
is double-checked by two persons.
Thawing of the straws.
The straw is removed from the freezing container using a pincet. The end with the steel ball is
cut off approximately 1 cm. from the end. The straw is placed with the open end down, in a
mini-test-tube for 15 minutes at room temperature.
The coloured flag end is then cut off and the sperm drips out into the test-tube.
The straw is then cancelled in the freezing protocol and donor ID etc. noted in the patient's
chart.
Evaluation of the frozen - thawed sperm.
1. The thawed sperm sample is pulled through a pipette 3 times, to homogenise the sample.
2. A droplet is placed in the Makler chamber and observed under the microscope.
3. All counts are noted in the patient's chart and in the freezing protocol.
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21. Freezing of sperm.
Straws to be used are selected by colour and all the patient's ID etc. are attached to the straw.
Also in the patient's chart, the straw colour and number are noted before being placed in the
freezing container .
1. The fresh sample is viewed under the microscope and the count and motility is noted.
2. All samples with white blood cells (leuc.) shall not be frozen, as the leucocytes will disintegrate
and harm the sperm during freezing.
3. The freezer is prepared with liquid Nitrogen etc. depending on the type of machinery to be
used.
4. The freezing medium is taken from 50C to room temperature. The freezing medium is added
to the sperm sample thus 0.4 ml freezing medium per 1 ml sperm and mixed.
5. The straw takes up to 0.5 ml. Thus 0.5 ml is applied to every straw .
6. The freezing curve has to be defined as follows: -2 0C/minutes until -800C.
7. The straws are then placed in the freezing container using a pincet which has already been
cooled before using it on the straws.
Safety procedures – freezing:
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22.1 Cleaning
Daily. Wash the floor before 8.00 by cleaning staff.
Lab in the afternoon: Wash tables and LAF with a 5-10% Detergent 7x soap solution. Clean with
distilled water, with 70% ethanol and distilled water.
Wash stands, racks, warming blocks with 70% Ethanol and distilled water.
Weekly. Afternoon. Wash microscopes and centrifuges with the 5-10% detergent soapsolution.
Wash with distilled water, clean with 70% Ethanol and distilled water.
Monthly. Afternoon. Wash all surfaces with 5-10% Detergent 7X solution. Wash with distilled
water, clean with 70% ethanol and distilled water.
Main cleaning in June. Empty all drawers, cupboards and shelves. The main cleaning is done
by the cleaning assistants together with the technicians.
Cleaning of the heating cabinets, centrifuges and incubators are done by the technicians.
22.2 Cleaning of the LAF bench
1. During cleaning of the LAF-bench put the flow on maximum.
2. Everything that can be removed from the LAF shall be removed.
3. 7 X is used for washing everything.
4. Sterile water is used for removing all 7X.
5. 70% Ethanol is used for final disinfection and distilled water.
Everything is then replaced in the LAF.
22.3 Cleaning of the incubator
June: Wash the incubator with a 5-10% Detergent 7X solution. Wash with distilled water, clean
with 70% ethanol and distilled water. Let the door open when not in use.
August: Wash with distilled water
December: Wash wih distilled water
23. Disposal
23.1 Biological material
Biological material and media are disposed through the hospita's system for biological
material. Describe.
23.2 Syringes and pipettes
Syringes, glasses, pipettes and catheters shall be disposed through the “Sharpsafe” system.
Describe.
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24.0 Quality Assurance
Utensils
Keep track of the lot no, date and treatment no. Scheme?
Keep the information for 5 years.
Media
Keep track of the lot. nr., date and treatment nr. Scheme?
Keep the information for 5 years.
Incubator
Weekly. Control the pH and PCO2.
The day before control, place 1 Petri-dish containing IVF-media in the incubator. The analyse of
the IVF-media is done in an ABL. The results shall be in the following areas.
PH
7,36 – 7,43
PCO2 4,4 – 5,6
Note the results on the scheme. Keep the results for 5 years.
If the results are not in the accepted areas, adjust the incubator and check again the next day.
Weekly. Change the water in the bottom of the incubator. Use pyrogen free distilled water. Note
the date on the scheme.
Temperatur. Every day. Check Temperature.
Every 3 months. Check temperature with external equipment.
The LAF bench
Service once a year. New HEPA filter. Note the service and sign by the service company.
The water heater is continuously filled with water. Change the water once every month. The
tubes, once a year.
Heating table
Check once every 3 months with external equipment.
Incubator
Cleaning and Disinfection
Cleaning
The exterior of the incubator should be kept clean by wiping over with a soft cloth and
soapy water.
Disinfection
1.
The recommended disinfecting agent for use with the incubator is Virkon.
Chemical specifications and supplies of this product can be obtained from
your distributor. There will be no adverse effects to the incubator if the
following procedures are carried out:
2.
Make a solution of 1% Virkon in water. NOTE: Gloves should be worn as a
routine precaution.
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3.
Switch of the incubator and disconnect from the main s supply.
4.
Position the black cover over the CO2 sensor.
5.
Dampen a clean cloth with Virkon solution and wipe down all external
surfaces. Take care not to let any of the liquid come into contact with any
electrical outlets or assemblies.
6.
Note: It is very important to ensure that no disinfectant solution is spilled onto
the brass CO2 sensor at the rear of the chamber. Failure to use the protective
cover may result in damage to the sensor which may effect your warranty.
7.
Remove all shelves, runners, supports and the humidity reservoir.
8.
Wipe down the inside of the chamber with Virkon solution.
9.
Place the internal components of the chamber in a 1% solution of Virkon.
Leave to soak for 10 min before wiping off the excess liquid. Re-assemble in
reverse order before switching the incubator on.
10.
Never use the following substances to clean the stainless steel as damage will
result: Sodium Azide, Aqua Regia, Iodine, Ferric Cloride, Sulphuric Acid.
11.
Do not leave water in the humidity tray if the incubator is switche of or a high
temperature decontamination cycle is started, as this may result in damage to
the CO2 sensor.
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