Intracytoplasmic Sperm Injection (ICSI)-Procedure And Equipment

Monday, August 04, 2008

Harald Andrulat and Sonja Voss, Eppendorf AG, Hamburg, Germany

Abstract
The number of infertile couples has increased in recent years and is now estimated at 15 – 20 % of all couples worldwide. Intracytoplasmic sperm injection (ICSI) has become a powerful means of overcoming infertility in azoospermic patients. With the development of ICSI, the modern infertility lab typically includes micromanipulation equipment attached to inverted microscopes. This Application Note focuses on the microinjection procedure itself.

Introduction

Since its introduction into reproductive medicine in 1978 [1], in vitro fertilization has been considered a successful method of treating tubal sterility. Almost one decade later, micromanipulation techniques were first applied successfully to the treatment of male-factor infertility [2]. It has been estimated that approximately 40 % of sterility in couples can be attributed to male subfertility. ICSI has raised hopes that these couples can have children of their own. This method of treating predominantly male-factor patients has achieved a breakthrough [3], and it has established itself as the preferred method of treatment in the field of assisted reproduction. ICSI obtains an oocyte via follicle aspiration, removal of the cumulus cells and corona radiata and the preparation of semen in accordance with the Mini Swim-Up process [4] or other preparation procedures [5]. Subsequently, one sperm is injected into this oocyte through a thin glass capillary (pipette). If fertilization occurs, then the embryo is transferred into the uterus 48 hours after microinjection (for further review see [6]).

In general, the Eppendorf system has a number of special features. The storage of up to three positions helps to speed up the procedure. Therefore the time needed for ICSI of five oocytes is less when using the Eppendorf system as compared with a conventional ICSI system [7].

Materials and methods
Devices
• Inverted microscope equipped with Hoffmann^® Modulation Contrast or Differential Interference Contrast (DIC) and 10 x, 20 x and 40 x objectives
• 2 TransferMan^® NK2 micromanipulators (one for moving the holding capillary and another for transferring the sperm)
• Adapter for inverted microscope
• CellTram^® Air microinjector for holding the oocyte
• CellTram^® vario microinjector for transferring the sperm

Consumables and media
• Light mineral oil (e.g., M-8410 (Sigma))
• Shallow Petri dishes, tissue-culture-grade (e.g., 351006 Petri Dishes (BD Falcon))
• VacuTips holding capillaries
• TransferTips^® (ICSI) injection capillaries
• Culture media (HEPES-buffered, supplemented with antibiotics, protein and pyruvate)

Microinjection dish preparation
It is essential to heat all media and oil to 37° C prior to use. For ICSI, place one droplet of PVP and several droplets of medium (10 µl – 50 µl) in the center of a Petri dish. Two larger droplets (50 µl – 200 µl) may also be needed for the storage of spermatozoa and/or equilibration of the capillaries. Then the droplets are completely covered with light mineral oil to maintain the stability of droplets as well as the temperature and osmolarity. Once prepared, the microinjection dish may be placed into the incubator until required.

Preparation of the microinjection capillaries
The microcapillaries have to be fitted, aligned and equilibrated before starting the ICSI procedure. Since the holding pipette is much bigger than the injection pipette, it can be used as a guide for positioning and equilibrating. First, the micropipettes have to be fitted into the universal capillary holder, which is connected to the microinjector via a tube. When working with oil-filled systems (e.g., CellTram vario), please make sure that no air bubbles are in the system. Gently push the capillaries past the sealing rings inside the tool holder.

After attaching the universal capillary holder to the the X head of the TransferMan NK2, check the alignment. The injection angle can be adjusted independently via the knurled screws and the angle mark on the X head (Fig. 3).

To align the capillary in the vertical position, the universal capillary holder can be rotated, even when the pipette is tightly gripped in place. Both pipettes have to appear straight in the field of view (Fig. 4).

The alignment in the horizontal plane has to be done with great care. The following points must be taken into account: the holding pipette has to be aligned without tilt, as it needs to lie flat on the bottom of the dish in order to aspirate the oocyte in a controlled manner. In contrast, the injection pipette needs to tilt downwards slightly so that the tail of the spermatozoon can be broken properly.

It is also necessary to prime micropipettes with medium before use so that the manipulated gametes never come into contact with air or oil. Usually, equilibration is achieved using ICSI media.

Storing of positions
The TransferMan NK 2 can store up to 3 positions. The capillary can be moved easily in any direction (x/y/z) by means of a joystick. By pressing the joystick button twice (double-click) the capillary can be returned to a preset position. Usually one position is set as a “parked” position, while another one serves as the “working” position.

Procedure (Fig. 5): A “working” position in the focal plane is chosen for the holding side (position 1 H) as well as for the injection side (position 1 T). The transfer capillary and the holding capillary are directed at the focal plane, and the positions are stored as Pos. 1 (1T and 1H). In addition a “parked” position for both capillaries is stored slightly above the droplet so that the pipettes do not interfere with the gametes as the dish is moved around the stage. The positions in the overlay medium are defined as Pos. 2 (2T and 2H)

Microinjection
Load the sperms into the PVP drop. Place each oocyte into one of the medium drops. Press the joystick button twice to lower the TransferTip (ICSI) capillary to position 1 T. At 200x magnification immobilize a sperm cell either by a quick movement of the TransferTip (ICSI) capillary via the tail or by pressing the tail of the sperm cell against the bottom of the dish (Fig. 6).

Aspirate the sperm cell, tail-first, into the TransferTip (ICSI) capillary as flatly as possible by rotating the knob of the CellTram vario. Press the joystick button twice to move the transfer capillary which now contains the sperm cell up into the overlay medium (2T). Move the Petri dish until you can see an oocyte in one of the surrounding drops and bring it into focus. Press the joystick button of the second TransferMan NK 2 to move the holding capillary from position 2 H to position 1 H. The oocyte is attached gently but firmly to the holding capillary with the help of negative pressure created by the CellTram Air device. The first polar body should be in the 6 o’clock or 12 o’clock position; therefore you may need to turn the oocyte with the help of the TransferTip (ICSI) capillary which has been lowered again to position 1T until the polar body comes to rest in either position. This requires the negative pressure on the CellTram Air to be slightly varied.

Then sharply focus on the TransferTip (ICSI) and the oocyte, move the spermatozoon along the capillary and bring it to rest at its very tip by rotating the knob of the CellTram vario. By moving the joystick slightly, carefully push the transfer capillary through the zona pellucida and the oolemma into the ooplasm at 3 o’clock.

The oocyte should be pricked in the middle so that the oolema membrane is gently and atraumatically broken. Advance the injection pipette to “suck up” the ooplasma into the injection capillary to be sure that the membrane is ruptured. Deposit the aspirated ooplasm and the spermatozoon towards the center of the oocyte (Fig 7). To introduce a minimal volume of the medium and PVP solution into the cytoplasm, gently withdraw the transfer capillary after the head of the sperm cell has left the pipette tip. Release the injected oocyte from the holding capillary and move both capillaries to position 2 by pressing the joystick button twice.

If several oocytes are obtained, only 3-4 oocytes are injected as a rule. They are placed into Ham’s F- 10-medium [4] and the remaining oocytes are then injected.

Assessment of fertilization
About 16 - 18 h after microinjection check the oocytes for the presence of pronuclei and polar bodies. After another 24 h, score the embryos (e.g., equal size of blastomeres) and transfer them into the uterus.

References
[1] Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet 1978 Aug 12;2(8085):366.
[2] Lanzendorf SE, Maloney MK, Veeck LL, Slusser J, Hodgen GD, Rosenwaks Z. A preclinical evaluation of pronuclear for- mation by microinjection of human spermatozoa into human oocytes. Fertil Steril 1988 May;49(5):835-42.
[3] Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992 Jul 4;340(8810):17-8.
[4] Al-Hasani S, Kupker W, Baschat AA, Sturm R, Bauer O, Diedrich C, et al. Mini-swim-up: a new technique of sperm prepa- ration for intracytoplasmic sperm injection. J Assist Reprod Genet 1995 Aug;12(7):428-33.
[5] Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intra- cytoplasmic sperm injection. Hum Reprod 1993 Jul;8(7):1061-6.
[6] Fleming SD, King RS. Micromanipulation in Assisted Conception. Cambridge: Cambridge University Press; 2003
[7] Safaa Al-Hasani, Otmar Bauer, Michael Ludwig, Rita Sturm, Oya Karabulut, D.Kahle, et al. Results of intracytoplasmic sperm injection using the microprocessor controlled TransferMan Eppendorf manipulator system. Middle East Fertility Society Journal 1999;4(1):41-4.

We would like to thank Prof. Dr. S. Al Hasani, Department of Obstetrics and Gynecology , Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany, for his support and positive assessment of our New Manipulator Generation for ICSI.