Intracytoplasmic Sperm Injection (ICSI)

Technical Guide Intracytoplasmic Sperm Injection (ICSI) Technical Guide Eppendorf Cell Technology

Eppendorf Cell Technology Intracytoplasmic sperm injection Introduction The number of infertile couples has increased in recent years, and it is estimated that 15% to 20% of all couples worldwide face infertility issues. Since its introduction into reproductive medicine in 1978, 1 in vitro fertilization has been considered a successful method of treating tubal sterility, and after years of research it is establishing itself as the preferred method of treatment in the field of assisted reproduction. 2 With increasingly intensive diagnoses of both partners, certain aspects of male subfertility are coming to the forefront; currently, almost 50% of infertility in couples can be attributed to male subfertility. 3 Intracytoplasmic sperm injection (ICSI), one method of treating male subfertility, has achieved a breakthrough 4, and it is gaining in importance, raising hopes that these couples can have children of their own. Preparation for ICSI involves obtaining an oocyte by means of follicle aspiration, removing the cumulus cells and corona radiata, and preparing the semen in accordance with the Mini Swim-Up process 5 or other preparation procedures. 6 One sperm is then injected into the oocyte with a thin glass capillary (pipette). If fertilization occurs, the embryo is transferred into the uterus 48 hours after microinjection. For the microinjection process described, Eppendorf offers ideal tools for easing the workload: the TransferMan NK 2 micromanipulator (Fig. 1) is used for moving the holding capillary, and a second TransferMan NK 2 is used for transferring the sperm; CellTram Air (Fig. 2) holds the oocyte; CellTram Oil (Fig. 3) transfers the sperm. In conjunction with an inverted microscope and Hoffman Modulation Contrast or DIC (differential interference contrast) optics, microinjection can be performed quickly and easily. With four easyto-understand buttons, the TransferMan NK 2 is simple to operate, and for every TransferMan NK 2, two spatial coordinates are defined and stored. Fig. 2 CellTram Air Fig. 1 TransferMan NK 2 micromanipulator Fig. 3 CellTram Oil

Procedure Instrument Set-up The capillary is easily moved in any direction (x, y and z) by means of a joystick. By simply pressing the joystick button, it can repeatedly assume one of the preprogrammed positions. A point in the focal plane is chosen as the oocyte holding side (Fig. 4, Position 1 H), where the sperm can be easily injected with the second TransferMan NK 2 (Position 1 T). For Position 2, it is recommended that the manipulators and capillaries use a point outside of the drops in the overlay oil. Holding side (H) Position 2 H Transfer side (T) Position 2 T Sperm Set-up Magnified 200x, a sperm cell is immobilized by quickly using a transfer capillary to press the tail of the sperm cell against the bottom of the dish until it stops moving. The sperm cell is aspirated tail-first into the transfer capillary by rotating the knob of the CellTram Oil or CellTram vario. By pressing the joystick button, the transfer capillary, which now contains the sperm cell, moves into the overlay oil. The petri dish is moved until an oocyte in one of the drops surrounding the sperm medium is visible, then the oocyte is brought into focus. The oocyte is held by the holding capillary (inner diameter approximately 20 µm) of the second TransferMan NK 2 and the simultaneous rotation of the knob of the CellTram Air in Position 1 H. Position 1 H Position 1 T Fig. 4 Schematic view of injection into oocyte The transfer capillary and the holding capillary are directed at the focal plane, and the positions are stored as Position 1 (T and H). Similarly, the positions in the overlay oil are defined as Position 2 (T and H). The sperm and ova are put in several drops of oil onto the petri dish. Pressing the joystick button lowers the transfer capillary (inner diameter approximately 4 µm to 5 µm) to Position 1 T. Injection into oocyte Oocyte Set-up Magnified 100x, the oocyte is turned by the transfer capillary, which has been lowered again (Position 1 T) until the polar body comes to rest either at the 12 o clock or 6 o clock position (the underpressure on the CellTram Air will vary slightly). 3

Procedure a Injection In this position, the holding capillary keeps the oocyte in place with the CellTram Air (d) (turn the knob to adjust holding pressure). Keeping the capillaries and the oocyte (magnified 400x) in sharp focus, the sperm cell in the transfer capillary is then brought to the tip by adjusting the pressure of the CellTram Oil (b) or CellTram vario (c). b c The joystick is moved slightly to push the transfer capillary carefully through the zona pellucida and the oolemma into the ooplasm at the 3 o clock position. The oocyte should be pricked in the middle so that the oolemma membrane is penetrated gently and atraumatically. If this is not immediately successful, continue to aspirate until the membrane perforates the sperm cell is then injected. In order to introduce little to none of the medium and PVP solution into the cytoplasm, the transfer capillary is gently withdrawn after the head of the sperm cell has left the capillary glass tip and the transfer capillary is no longer inserted. The injected oocyte is released from the holding capillary and both capillaries are moved to Position 2 by pressing the joystick button. As a rule, if several oocytes are obtained, only 3 to 4 oocytes are injected, placed into Ham s F-10-medium, 5 and then the remaining oocytes injected. Further methods of procedure are described in Al Hasani, et al. 5 e d Eppendorf equipment for an optimal workstation Manipulation 2 x TransferMan NK 2 micromanipulators (including microscope adapter) [a] Injection Holding 1 x CellTram Oil [b] or, alternatively, 1 x CellTram vario [c], TransferTip -RP [e] 1 x CellTram Air [d], or, alternatively, 1 x CellTram Oil [b] 4

Eppendorf precision accessories TwinTip-Holder The new TwinTip-Holder* NMG (Fig. 5) provides individual control over two microtools using only one micromanipulator. The patented swivel mechanism of the TwinTip-Holder NMG and the electronic control of the TransferMan NK 2 allow the work and parking positions for both capillaries to be stored and then recalled instantly. This feature prevents the parked capillary from damaging either the sample material or the tool in the work position. The rapid, safe and reproducible exchange of the work positions of both tools protects test material, reduces manipulation times and increases efficiency levels. Fig. 5 TwinTip-Holder NMG Typical applications include assisted hatching and subsequent blastomere biopsy. The complete workstation for these applications consists of an inverted microscope, one TransferMan NK 2 micromanipulator, one TwinTip-Holder, one CellTram Air pressure dispenser for holding purposes, and two CellTram Oil or CellTram vario pressure dispensers for manipulating the sample material. Microcapillaries To reiterate, ICSI has rapidly developed into one of the most important in vitro fertilization techniques for male subfertility. Continuous advancement in methods and technology ensures a high success rate, even in the most difficult cases. Success is only possible by protecting the sensitive cells involved in the fertilization process. This requires well-designed capillaries of a consistently high quality. TransferTip and VacuTip have been developed to meet these requirements. The consistent tip diameter and well-defined shape of the TransferTip enable the sperm to be transferred easily into the oocyte. The VacuTip holding capillary fixes the oocyte firmly and safely in place for optimal injection. Eppendorf precision microcapillaries are gammasterilized to avoid use of cytotoxic sterilization reagents such as ethylene oxide. They are also tested by an independent institute via mouse embryo development assay to ensure their safety. Choose from three types of TransferTips (TransferTip-F, TransferTip-RP and TransferTip-R) to best suit your ICSI application. All Eppendorf microcapillaries are delivered in Capillary Safe (Fig. 6) transport packaging to ensure worry-free handling and protection for users (see ordering information, page 8). *U.S. Pat. 6,159,199. References: 1. Steptoe PC, Edwards RG. Birth after reimplantation of a human embryo. Lancet. 1978;ii:366. 2. Van Steirteghem AC, et al. Higher success rate by intracytoplasmic sperm injection than subzonal insemination. A report of a second series of 300 consecutive treatment cycles. Hum Reprod. 1993;8:1055 1060. 3. Küpker W, Al Hasani S, Bauer O, Diedrich K. New technologies of assisted fertilization. Fertilität. 1994;216 220. 4. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into oocyte. Lancet. 1992;340:17 18. 5. Al Hasani, S, et al. Intracytoplasmic sperm injection using the Mini Swim-Up process. Fertility. 1995;11:111 114. 6. Van Steirteghem AC, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod. 1993;8:1055-1060. Fig. 6 Capillary Safe

Precision capillaries for ICSI Product features in common TransferTip (ICSI) l Glass microcapillaries for human sperm injection and the containment of l Sterilized by validated gamma irradiation l Noncytotoxic For sperm injection using the ICSI technique l 4 µm inner diameter l 7 µm outer diameter l Heat-formed spike human egg cells l 1 mm outer diameter (continuous testing with mouse embryo test) eases penetration l 35 tip angle VacuTip For holding suspension cells (e.g., oocytes or blastocysts) l 15 µm inner diameter l 100 µm outer diameter l 35 tip angle l TransferTip-F, flexible flange, 0.4 mm long l VacuTip To supply you with the ideal capillary for your personal work methods, we offer three types of TransferTips. They are differentiated by the length and shape of the angled flange. l TransferTip-RP, rigid, parallel flange, 0.5 mm long l TransferTip-R, rigid flange, 1 mm long 6

Technical specifications Eppendorf TransferMan NK 2 Control of axes: Operating range: Resolution: Drive: Max. working speed of instrument: X, Y, Z via central joystick; proportional kinetics X, Y, Z >20 mm each X, Y, Z, approx. 40 nm per microstep Disc stepper motor, 40 nm per microstep 7,500 µm/s Work radius Coarse: User-defined from 12,500 µm to 5 µm, in steps of 5 µm Fine: approx. 0 µm to 1.5 µm Capillary exchange: Position storage: User profiles: Weight: Module unit, X, Y, Z complete: Control panel with power supply: Control panel dimensions (W x D x H): Power supply: Via programmable Home function 3 independent positions can be stored and recalled for all 3 space coordinates 4 independent profiles can be stored and recalled 3.1 lb/1.4 kg 6.0 lb/2.7 kg 10.63 x 8.07 x 6.30 in/27.0 x 20.5 x 16.0 cm 120 V 240 V/50 Hz 60 Hz Eppendorf CellTram microinjectors CellTram Air CellTram Oil CellTram vario Generation of pressure: Cylinder/piston system Cylinder/piston system Cylinder/piston system air-filled, maintenance-free oil-filled, maintenance-free with gears, oil-filled, maintenance-free Diameter of dial: 40 mm 42 mm/34 mm (coarse/fine) Volume change per revolution: 88 µl 9.8 µl 9.6 µl/960 nl (coarse/fine) Total volume that can be set: 2.640 µl 980 µl 960 µl Total piston stroke: 15 mm 50 mm (corresponds to 30 revolutions) (corresponds to 100 revolutions) Minimum volume that can be set: <200 nl <20 nl <20 nl/<2 nl (coarse/fine) Maximum pressure: 2,900 hpa 20,000 hpa Pressure tube: Length: 1 m Diameter: 1 mm (inner) Material: polyethylene Oil filling: Not applicable Approx. 2 ml total volume Oil: embryo-tested

Ordering information Description Applications Catalog No. TransferMan NK 2 920000011 Prime Tech Piezo Impact Drill 920002307 Instruments/accessories Adapters, for all commonly used microscopes available upon request TwinTip-Holder, twin capillary holder For TransferMan NK 2 920007767 Universal capillary holders For glass capillaries and grip heads 920007392 CellTram Air, manual pneumatic pressure-control system For holding cells 920002021 CellTram Oil, manual hydraulic pressure-control system For transferring cells or for manual microinjection 920002030 CellTram vario, manual hydraulic microinjector, For transferring cells or for manual microinjection with gears 1:1 and 1:10 920002111 Consumables VacuTip, sterile glass capillaries, 25 pcs. For holding large cells (e.g., oocytes) 930001015 TransferTip-F (ICSI), sterile glass capillaries, For injecting sperm into an oocyte (ICSI) with flexible flange, 25 pcs. 930001031 TransferTip-R (ICSI), sterile glass capillaries, For injecting sperm into an oocyte (ICSI) with rigid flange, 25 pcs. 930001066 TransferTip-RP (ICSI), sterile glass capillaries, For injecting sperm into an oocyte (ICSI) with rigid, parallel flange, 25 pcs. 930001074 For more information, contact your Eppendorf North America Cell Biology Sales Specialist at 800-645-3050. 8301-C203E-07 2006 Eppendorf AG. Eppendorf, TransferMan, TransferTip, and CellTram are registered trademarks of Eppendorf AG. Hoffman Modulation Contrast is a registered trademark of Modulation Optics, Inc. Product appearance, specifications, and/or prices are subject to change without notice. www.eppendorf.com Email: info@eppendorf.com Application hotline: 516-515-2258 In the U.S.: Eppendorf North America 800-645-3050 In Canada: Eppendorf Canada Ltd. 800-263-8715