ICSI TECHNIQUE

1. Oocyte preparation for ICSI

Oocytes are collected in a falcon tube (3037), previously heated, and the follicular liquid is immediately sent to the embryology laboratory, where through the use of stereomicroscope, they will be identified and then classified. After identificated, oocytes are placed in a 1.0 ml hyaluronidase 80 UI/ml (Hyase 1) solution, for 30 seconds, making na up and down movement with the pasteur pipette. Soon afterwards, they are washed many times in na IVF 50 medium and placed in a petri dish with 50 µl IVF 50 culture medium microdrops and covered with previously equilibrated mineral oil (Ovoil 150).

Then they are incubated in a heater for at least 20 minutes until oocyte denudation time (cleaning out the cumulus). At the end of the incubation period the cumulus-corona complex is mechanically removed by a pipette coupled to a pipetter (Stripper). This pipette must have a 125 caliber µm. Oocytes are classified in metaphase II (mature oocytes with the presence of the first polar body), metaphase I (immature oocytes with na absence of polar body), prophase I (immature oocytes with the presence of germinative vesicule), atretic (oocytes presenting degeneration process) and fractured (oocytes with cytoplasm or zona pellucida damage). This classification is made by the denudated oocytes observation through na inverted microscope (Nikon-Eclipse) with 200X to 400X magnification. Injection is performed in metaphase II oocytes (MII) until 4 hours after retrieval. Metaphase I oocytes (MI) remain in culture until extrusion of the first polar body (4 to 6 hours after retrieval).

2. Semen preparation for ICSI

Information on the seminal quality are provided by the andrology laboratory at the first spermogram procedure, preceding the treatment cycle. On the oocyte aspiration day semen is collected through masturbation, in the absence of azoospermy, and immediately sent to the embryology laboratory, for sample liquefaction and ICSI preparation.

Samples considered to be normal (concentration > 20 x 106/ml; motility > 50%; normal morphology > 14% - Kruger) or moderately oligospermic (concentration 6 to 19 x 106/ml) are processed in the following fashion:

1. To each 1.0 ml seminal sample is added 6.0 ml Sperm Rinse Medium.

2. Centrifuge at 1500 rpm for 10 minutes.

3. Remove supernatant and ressuspend remaining pellet with 1.0ml Sperm Rinse.

4. Centrifuge for 5 minutes more.

5. Remove supernatant and ressuspend pellet with about 1.0ml Sperm Rinse (depending on the spermatozoon concentration).

Samples with severe oligospermy (concenration < 5 x 106 /ml) are processed in the following fashion:

1. To the total sample volume 5.0 ml Sperm Rinse is added.

2. Centrifuge at 1500 rpm for 10 minutes.

3. Remove supernatant and ressuspend pellet with 0.5 to 0.8 ml Sperm Rinse.

Samples removed from the epididym (PESA) with with normal spermatozoon concentration receive the same treatment as the normal semen proceding from na ejaculation. If the sample has low sperm concentration, only one rinse is made.

Samples extracted from the testicle by TESA are inspected for the presence of spermatozoa, placing the syringe content on a petri dish with Gamete 100 drops having each of them about 10 µl, covering them with mineral oil and observing them through the inverted miocroscope at 200X magnification. If the sample shows sparse spermatozoa, they must be removed from the droplets and directly transferred to the PVP where they will be ready for injection. Samples obtained by TESE are processed in the following way: Tissue removed from the testicle must be placed on a petri dish without center, with 2.0 ml Gamete 100 medium and sent to tha ICSI lab, for dissection on glass lamines. The sample is checked for the presence of spermatozoa soon after dissection. If there are spermatozoa, the dish content is placed inside a conic tube and incubated until injection time. The tisue is then discarded and the supernatant is centifuged at 3000 rpm for 5 minutes. After centrifugation the supernatant is removed and the pellet ressuspended with 0.5 ml Sperm Rinse. All samples after capacitation must be placed in the incubator until injection time at 37oC and 5% CO2 concentration ( see more detail about these procedures ).

3. Preparing for microinjection

In a Falcon Petri Dish (1006) one central 5 ml droplet ICSI 100 is placed (to immobilize spermatozoa) surrounded by 8 5ml Gamete injection medium droplets, covered by OVOIL 150 mineral oil. In ICSI 100 is placed 1-2ml already capacitated seminal sample, and in the surrounding droplets 1 oocyte per droplet.

The ICSI procedure is performed on a heating platina at 37oC coupled to the inverted microscope (Nikon-Eclipse) at 200x – 400x magnification, using Hoffman’s phase contrast. The microscope is equipped with a videocamera system and video monitor, by which the procedure can be observed.

One only spermatozoon is selected and mechanically immobilized by a light pressure over its tail. The tail is then aspirated into the injection pipette.

With a holding pipette, the first polar body is placed at the 6 o’clock position (depending on the injection needle opening). The oocyte is then fixated by a gentle suction in the holding pipette. Only one spermatozoon, immobilized inside the injection pipette, is injected at the 3 o’clock position. After the injection of the pipette into the ooplasm, a small portion is suctioned to ascertin the rupture in the cytoplasmic membrane and, soon after this rupture, the spermatozoon is delivered together with the ooplasm.

After the injection, oocytes are washed in IVF 50 medium and placed in a Petri Dish with Falcon center (3037) containing 1 ml IVF 50 medium covered by mineral oil and placed in the incubator until verification of fertilization.

4. Verification of Fertilization

Verification of fertilization is done between 16-18 hours after ICSI, through the inverted microscope at 400x magnification. The presence of pronuclei and polar bodies is checked, as well as the presence of abnormalities in fertilization (absence of pronuclei, more than two pronuclei, degeneration, etc.).

Fertilization is considered to be normal when two pronuclei can be seen, containing nucleoles inside. If only one pronucleus is seen, a second checking id made after 4 hours. When there are three or more pronuclei, the embryo is immediately discarded.

Fertilized oocytes are washed in G1.2 medium and grouped 2 by 2 in petri dishes without center, in 50ml G1.2 medium droplets, covered by mineral oil. Dishes are again placed in the incubator and remain in this same culture medium until 72 hours after ICSI (day 3). It is not necessary to acces embryos before 48 hours, thus avoiding the constant opening of the incubator, which could bring damage to the cultures.

In case transfer is performed on day 3 (72 hours), the embryos must remain in IVF 50 culture medium until 48 hours and then the medium must be changed to complete the remaining time (72 hous).

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