Standard Operating Procedure IVF and ICSI procedures

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1 Pages: 1 of 6 PRINCIPLES Oocytes are aspirated from mature Graafian follicles after the administration of ovarian hyperstimulation pharmacological regimes designed to produce multiple follicular development. Oocytes must be aspirated and maintained in an environment that maintains the temperature at 37 C and the fluid ph (in this case follicular fluid) at A small change from physiological in these delicate cells can eliminate the potential of this material to develop and produce live births. Background Originally, oocytes were removed by laparotomy, quickly superceded by laparoscopy techniques. A great breakthrough in oocyte retrieval techniques was made by the development of ultrasoundguided vaginal oocyte retrieval techniques: Usually, the oocyte retrieval procedure is performed in the operating theatre under anaesthesia. Oocytes are temporarily stored at 37 C in follicular fluid for transport to the laboratory. Equipment required All equipment required is listed in attachment I (equipment required) and II (consumables required) PROCEDURE Preparation All culture media/oil etc is conserved at 4 C and discarded upon expiry. 12 hours prior to use, dishes for preparation, treatment and insemination of oocytes are prepared under oil and allowed to incubate in an atmosphere of 37 C, 6% CO 2. It is suggested that a minimum quantity of culture material includes: 1) 1 x pick-up dish containing 4ml fertilisation medium overlaid with 2ml oil 2) 1 x dish for culture of oocytes containing 5-6 drops (50ul) fertilisation medium overlaid with 5ml oil per 10 oocytes predicted. 3) 1 x hyaluronidase dish containing 1 drop (50ul hyaluronidase 80IU/ml) marked and 4-5 drops fertilisation medium (50ul) overlaid with 5ml oil per 10 oocytes predicted. 4) 1 x dish for culture with numbered 50ul drops (5-6 per dish) of fertilisation medium. Prepare 1 dish for each 6 predicted oocytes. 5) 1 x dish containing 10ml oil for each patient.

2 Pages: 2 of 6 6) 1 x 10ml follicle wash medium to wash pick-up needle. Oocyte retrieval procedure After identification of the patient, the patient is anaesthetised and aspiration needle connected to the ultrasound probe. With ultrasound visualisation of the ovary, the needle is inserted into the follicle of interest. Aspiration ìs performed with a suction pump delivering mm Hg presure. Follicular fluid containing oocytes is collected into sterile Falcon tubes (2059). These are conserved at 37 C in follicular fluid briefly until the procedure is terminated and then rapidly transported to the laboratory. In the laboratory, oocytes are separated from the follicular fluid and any blood and other debris by examination under a dissection microscope with a 37 C heated stage and placed into a culture dish containing 4 ml of fertilisation medium overlaid with 2ml oil. Oocyte-cumulus complexes are counted, the clinician is informed, and these are placed into an incubator for culture until the material is required. It is recommended to culture for at least 2 hours prior to use. for insemination The procedure for insemination depends on the technique selected. Basically, 2 techniques are applied. In IVF, sperm and eggs are mixed and incubated together, whereas for ICSI, spermatozoa are injected into the cytoplasm of the oocyte, IVF ICSI 1) Prepare semen as described in SOP-LD-01 and dilute to final concentration 1 x 10 6 /ml. Place in incubator for at least 2 hours to enable equilibration. 2) Prepare insemination drops by diluting semen in numbered culture drops to a final concentration of 100,000/ml (20-30 sperm/field under 10 x objective). 3) Place a single cumulus-oocyte complex into each drop and incubate minimum 16 hours in an incubator. 4) Remove oocyte from cumulus by pipetting through a 140um bore pipette and examine for fertilisation. 5) Place into fresh, equilibrated culture medium suitable for embryo development and culture until transfer. 1) Remove cumulus from the oocyte by placing in equilibrated hyaluronidase. Oocytes can then be aspirated into a pipette 130uM diameter for removal of the theca interna.

3 Pages: 3 of 6 2) Cleaned oocytes are examined for maturity, immature oocytes separated from metaphase-ii stage oocytes and those suitable for insemination are cultured for minutes prior to use. 3) An ICSI dish (Falcon 1006) is prepared with a drop of PVP (7% solution) and several drops of HEPES-buffered culture medium (eg gamete buffer). 5ml equilibrated oil is overlaid to prevent drying. Dishes are equilibrated in an incubator at least 60 minutes prior to use. 4) Oocytes are placed 1 per drop of gamete buffer and sperm samples placed in the PVP drop. A single spermatozoa is injected into each oocyte by micromanipulation techniques. 5) Inseminated oocytes are then placed in an incubator in numbered culture drops for 16 hours before fertilisation check. Micromanipulation techniques Micromanipulation is performed using an inverted microscope equipped with Hoffmann Optics (10 x, 20 x and 40 x Objectives) and micromanipulators. Oil-filled microinjectors are used for the injection of the spermatozoa. Glass pipettes are used both to hold the oocyte (holding pipette) and inject the spermatozoa (ICSI pipette). These pipettes are positioned in parallel with the holding parallel to the microscope stage and the ICSI pipette angled with the tip slightly downwards (1-2 ). Spermatozoa are placed into the PVP drop and single sperm selected for ICSI. Selection criteria are: 1) Sperm motile or obviously living (i.e. in-situ movement) 2) Sperm of perfect morphology In the absence of these, sperm are selected by morphology nearest to the Kruger criteria. Selected spermatozoa are immobilised by touching the tail with the ICSI pipette just below the midpiece. The spermatozoa is then aspirated into the ICSI pipette. After blocking the oocyte with polar body at the 6 o'clock or 12 o'clock position, the ICSI pipette is advanced through the zona pellucida. Gentle suction is applied to rupture the oolemma, and then the spermatozoa is deposited in the cytoplasm of the oocyte. The ICSI pipette is then retrieved and the process repeated for each oocyte. When the process is terminated, oocytes are moved into numbered drops in a culture dish and placed in the incubator for culture. Fertilisation check At hours post insemination, cultured oocytes are cleaned (in the case of IVF) and examined for the presence of 2 pronuclei. Each oocyte is registered in number order. Fertilised oocytes are placed in culture medium suitable for cleavage of embryos in drops with the same number order. These are cultured until required for embryo transfer. Embryo evaluation and transfer

4 Pages: 4 of 6 On the day of transfer, embryos are analysed for rate of development, level of fragmentation, and quality using a quality score (De Placido et al., 2002). Embryos selected for transfer are placed together, photographed and replaced in the incubator until ready for replacement. Transfer procedure: 1) The patient and partner are again identified, the biological parameters explained, the number of embryos to be transferred agreed on, and the patient gives consent to the cryopreservation of any excess embryos. 2) Embryos to be transferred are shown to the patient through camera attached to a microscope. 3) Patient and clinician prepare for the transfer 4) When clinician is ready, embryos are aspirated into the transfer catheter and passed to the clinician. 5) The transfer catheter is checked for the absence of embryos after the procedure, as well as for the presence of blood. If blood is present inside the catheter, this is noted on the biological sheet. Where no embryos are present in the catheter post-transfer, the procedure is considered complete. 6) Where embryos are present within the catheter post transfer, the procedure must be repeated from point 4, and this noted on the biological sheet. 7) At the termination of the process, the patient is given a report signed by the biologist. Documentation All IVF/ICSI procedures should also be inserted into the database 'FLM'.. These should clearly state the patients' details, number of oocytes collected and used, a quality assessment of oocytes and embryos formed, number of embryos transferred and cryopreserved and the transfer details. ATTACHMENT I: Equipment required Incubator Gas regulator Carbon dioxide gas supply Emergency power supply for incubators 2 Test-tube warmers (dry thermoblock with inserts for 14ml Falcon tubes and 5ml Falcon tubes) Laminar flow hood with heated stage for oocyte manipulation (or see above) Or - Stereomicroscope ( Dissecting microscope ) for oocyte retrieval with heated stage + laminar flow hood Inverted microscope with heated stage Manipulator coarse + fine Anti vibration table for ICSI station

5 Pages: 5 of 6 Injectors Fridge Air filtration apparatus (CODA filter) Craft suction pump Portable incubator for oocyte retrieval (or thermoblock as above) Test-tube holders ATTACHMENT II: Consumables required Nunc delta surface small for oocyte/embryo culture Nunc delta surface large for excess oil Falcon 3004 p/u dish Falcon 1006 ICSI dish Falcon 2058 provette 6ml Falcon 2059 provette 14ml p/u Filtri Millex GS 0,22 m Eppendorf Biopur pipette tips Holding pipette Falcon 6551 sierologico 10ml Falcon 7543 sierologico 5ml ICSI pipette AZH pipette Falcon 2099 Falcon 7507 sierologico 2ml Falcon 7521 sierologico 1ml Bidistilled water Glass Pasteur pipette Syringe 1ml Alcohol lamp for pipettes with wick (or gas Bunsen burner) Thermometers C range Diamond-tip pencil for marking culture dishes Mouth pipette or foot pedal Powder free gloves Containers x sterilising pasteur Pipettman 20ul Electronic pipetter Bulbs x pasteur EtOH x burner Coverslips x TESA

6 Pages: 6 of 6 Sterile Gloves Speculum Transfer catheter P/u needle REFERENCES Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Lancet Jul 4;340(8810):17-8. Transvaginal sonographically controlled follicle puncture for oocyte retrieval. Dellenbach P, Nisand I, Moreau L, Feger B, Plumere C, Gerlinger P. Fertil Steril Nov;44(5): High outcome predictability after IVF using a combined score for zygote and embryo morphology and growth rate. De Placido G, Wilding M, Strina I, Alviggi E, Alviggi C, Mollo A, Varicchio MT, Tolino A, Schiattarella C, Dale B. Hum Reprod Sep;17(9): An oocyte score for use in assisted reproduction. Wilding M, Di Matteo L, D'Andretti S, Montanaro N, Capobianco C, Dale B. J Assist Reprod Genet Aug;24(8): Epub 2007 Jul 15.