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.
Embryo laboratories.
1. Preparation for oocyte pick-up
2. Oocyte pick-up
3. Insemination of the oocytes
4. Medium change
5. Embryo transfer
6. Embryo freezing
7. Thawing of embryos
8. Embryo destruction
9. 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.
- 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 LAF-
bench, in 250ml.Falcon flasks.
Storage: 2-8C.
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: 2-8C
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
1.3Preparation 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.
- 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
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 500l 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.
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: 2-8C.
30 ml flush medium is used per asp.
2.2Utensils:
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.
1. Approx. two hours after oocyte pick-up, will the oocytes be inseminated with purified and cleaned motile spermatozoa (see procedure 17).
- Check the ID Name and nr. of the sperm suspension.
- 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 500l IVF medium per well and 2 x 500l 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.
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 20l 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.
6. Embryo freezing.
Reagents:Embryo Freezing Medium4x10 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: 2-8C
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. 0030-001-
311.
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
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 500l 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
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: 2-8C
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: 2-8C.
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: 2-8C.
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. 0030-001-
311.
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.
The day before thawing embryos.
- The freezing journal and records are placed in the laboratories and 4-well dishes are marked with the colour and patient's ID.
- The wells are filled with the IVF medium.
The day of thawing.
- The media T1, T2, T3, IVF medium is placed in a 4-chamber well marked with
patient name and ID nr. (LAF Bench)
- At the freezing chart, it is confirmed which and where the straw to be thawed is placed. (see lab freezing file)
The thawing procedure.
- 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.
- The eggs are scored and left overnight in the incubator to cleave.
- The straws and eggs are all checked by two persons.
- 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.
- The next day : the state and score of the eggs after thawing.
- Before transfer : the state and score of the eggs
- Also how many eggs left in the freezer.
8. Embryo destruction.
- The patients’ eggs will, according to Danish law, be destroyed after 2 years.
- The embryos are identified, by means of the freezing journal. The day for destruction is noted in the lab file.
- 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.
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 pipette0.018 - 0.025
ICSI pipette0.0048 - 0.0056
Denuding pipette0,134 – 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.
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.
- 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.
- 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.
- 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-well dish for aspiration - see preparation for oocyte pick-up pg.3.
11.2: Preparation day.