Step-by-Step Guide to the ICSI Technique for Embryologists: Hands-On Training

Dr. Richika Sahay | 22 Aug 2024 | 19453 Views |

Introduction

The ICSI technique (Intracytoplasmic Sperm Injection) is a highly specialized micromanipulation procedure used in IVF labs worldwide. Mastery of this technique is crucial for embryologists aiming to optimize fertilization outcomes, particularly in cases of male infertility. This guide offers a detailed, step-by-step approach to performing ICSI, from sperm selection to embryo transfer.

1. Preparation of Equipment and Materials

Before starting the ICSI technique, ensure that all equipment is properly calibrated and that the following materials are prepared:

• Micromanipulator System: A high-precision micromanipulator connected to an inverted microscope.
• Microinjection Pipettes: Injection pipettes for sperm and holding pipettes for the oocytes.
• Culture Medium: Pre-equilibrated culture medium for sperm and oocytes.
• Incubators: To maintain the oocytes and embryos at the required temperature and gas environment.
• Sperm Sample: Prepared and processed sperm sample.
• Oocyte Dish: Dish containing the oocytes in a drop of culture medium, surrounded by mineral oil.
• Microtools: Glass needles for holding and injecting.

2. Sperm Preparation and Selection

• Sperm Processing: Begin by preparing the sperm sample using techniques such as density gradient centrifugation or swim-up to isolate motile sperm.
• Sperm Selection: Under the microscope, select a single, motile sperm with normal morphology. Use a denuding pipette to immobilize the selected sperm by applying a quick mechanical touch to the tail. This immobilization is crucial for successful injection.

3. Oocyte Preparation

• Oocyte Retrieval: After ovarian stimulation and egg retrieval, place the cumulus-oocyte complexes (COCs) in a culture dish.
• Denudation: Use a combination of hyaluronidase enzyme and mechanical pipetting to remove the cumulus cells surrounding the oocytes. This step allows better visualization of the oocyte’s polar body, which is essential for orientation during injection.

4. Setting Up the Micromanipulator

• Aligning the Pipettes: Mount the holding pipette on one side of the micromanipulator and the injection pipette on the other. The holding pipette should be slightly concave to gently secure the oocyte.
• Microscope Calibration: Ensure the microscope is correctly focused, and the magnification is set to allow clear visualization of the sperm and oocyte.
• Media Preparation: Add microdrops of pre-equilibrated culture medium under mineral oil in an injection dish. Place the oocytes in these drops.

5. Performing the ICSI Injection

1. Positioning the Oocyte: Using the micromanipulator, position the oocyte so that the polar body is at the 12 o’clock position, ensuring the metaphase plate is horizontal to avoid damaging the spindle.
2. Securing the Oocyte: Gently apply suction to the holding pipette to stabilize the oocyte.
3. Sperm Loading: Using the injection pipette, aspirate the selected sperm tail-first, with the head positioned near the tip of the pipette.
4. Sperm Injection: Penetrate the zona pellucida at a 3 o’clock position, move the pipette towards the center of the oocyte, and gently aspirate a small amount of ooplasm into the pipette to ensure the oocyte membrane has been breached.
5. Release the Sperm: Carefully expel the sperm into the ooplasm and slowly withdraw the injection pipette.
6. Release the Oocyte: Gently release the oocyte from the holding pipette, allowing it to rest in the culture medium.

6. Post-Injection Monitoring and Incubation

• Monitoring: Immediately after injection, observe the oocyte under the microscope for any signs of damage or abnormality. Discard any oocytes that appear lysed or damaged.
• Incubation: Place the injected oocytes back into the incubator for further development. Monitor for signs of fertilization, such as the appearance of pronuclei, within 16-20 hours post-injection.

7. Embryo Culture and Transfer

• Embryo Assessment: After fertilization, continue to culture the embryos until they reach the appropriate developmental stage (cleavage stage or blastocyst) for transfer.
• Embryo Transfer: Select the best quality embryos for transfer to the uterus. The remaining embryos can be cryopreserved for future use.

8. Documentation and Quality Control

• Record Keeping: Document each step of the ICSI process, including the number of oocytes injected, the fertilization rate, and any observations during the procedure.
• Quality Control: Regularly calibrate equipment and assess the quality of materials and culture media used in the procedure. Continuous training and assessment of embryologists’ technique are essential for maintaining high success rates.

Conclusion

Mastering the ICSI technique requires precision, practice, and attention to detail. By following these detailed steps, embryologists can enhance their skills and contribute to higher fertilization rates and successful IVF outcomes. Whether you’re new to the procedure or looking to refine your technique, this guide provides a solid foundation for performing ICSI with confidence and accuracy.

About The Author

Dr. Richika Sahay

MBBS (Gold Medalist), DNB (Obst & Gyne), MNAMS, MRCOG (London-UK), Fellow IVF, Fellow MAS, Infertility (IVF) Specialist & Gynae Laparoscopic surgeon,[Ex AIIMS & Sir Gangaram Hospital, New Delhi]. Read more about me