In Vitro Fertilization with ICSI

What should you know about IVF with ICSI? 

The implementation of ICSI Fertilization technique involves several ideally synchronized in the timeline steps:

1. Oocyte Retrieval (via follicle aspiration).
2. Oocyte Preparation.
3. Sperm Collection.
4. Surgical Sperm Retrieval PESA (Percutaneous Epididymal Sperm Aspiration); MESA (Micro Epididymal Sperm Aspiration); TESE (Testicular Sperm Extraction) or Micro–TESE (Microsurgical Testicular Sperm Extraction).
5. Sperm preparation.
6. Preparation of micromanipulation tools for ICSI (ICSI Injection Pipettes and ICSI Holding Pipettes preparation).
7. Compact stereomicroscope and other ICSI micromanipulation tools  preparation.
8. Oocyte immobilization, sperm immobilization for Intracytoplasmic Sperm Injection.
9. Intracytoplasmic Sperm Injection (one single premium–quality spermatozoon in injected in one oocyte).
10. Preparation of Assisted Hatching PZD (Zona Drilling Pipettes) for opening zona pellucida for Assisted Hatching of the tiny embryo (the micromanipulation tools that are vital for successful embryo implantation).
11. Biopsy micropipettes preparation for PGD (Blastomere Biopsy and Polar Body Biopsy).
12. Petri dishes preparation (after Embryo Creation, every embryo is transferred into the private embryo space).

Intracytoplasmic Sperm Injection in IVF cycle Bundle: how is it performed? 

IVF cycles are designed for effective coping with infertility challenges. Every IVF cycle has its exclusive Protocol with innovative medications, cutting–edge technological solutions, and exceptional techniques to manage the biological and genetic issues. It literally involves you in the other reality where everything is possible and everything has its own timeline. The opportunities are nearly limitless.

ICSI is performed in the embryology laboratory. The specialist who performs ICSI is called embryologist.

Micromanipulator for ICSI 

ICSI involves ideally synchronized in the timeline sequence of delicate microsurgical manipulations. These manipulations are performed under the Inverted Microscope for ICSI and IMSI, under In Vitro Fertilization Micromanipulation system with ICSI Micromanipulator or under Micromanipulator for ICSI.

Exclusively designed for micromanipulation, denuding and handling ICSI high–quality micropipettes are also prepared for covering every preference and every need of the embryologist.

ICSI Micropipettes what are they? 

ICSI micropipettes are used for the cutting–edge aspiration and injection the sperm directly into the oocyte’s cytoplasm. ICSI Injection Micropipettes and ICSI Holding Micropipettes of all sizes, shapes, and diameters are prepared for the management of every ICSI.

ICSI Injection Micropipettes for sperm

ICSI Injection Micropipettes with a short taper for added stability when injecting a single spermatozoon have a smaller inner diameter to minimize the impact on the oocyte. ICSI Injection Micropipettes with extra–long parallel taper, exclusively designed for very fine fluid control and suction during ICSI procedure with a sharp–blunt spike for easy penetration of the zona pellucida and gentle progression through the oolemma to minimize the risk of oocyte tearing.

High-quality ICSI Injection Micropipettes designed all sizes, shapes, and diameters enable accurate and precise manipulations.

Easy immobilization of sperm, very fine fluid control and suction during use, easy penetration of the zona pellucida and gentle progression through the oolemma with minimal risk of tearing, the optimal design for loading and injecting multiple sperms – ICSI Injection Micropipettes are designed to do wonders!

ICSI Holding Micropipettes for oocytes

Oocytes have their private ICSI “shuttles”. These “shuttles” are pipettes with a rounded tip and polished opening. ICSI Holding Pipettes are exclusively designed to accurately hold the oocyte (or embryo) flat and horizontal position during ICSI and other micromanipulation procedures.

Holding Pipettes “synchronized” with ICSI Injection Micropipettes literally make a breakthrough dual–micropipette ICSI system with depth control – the smartest and most powerful in IVF. Taken to the extreme, these micropipettes enable superfast fertilization. No other fertilization technique is like ICSI.

ICSI procedure how is it done? Notes that are left on the backstage

ICSI Devices what are they? The ICSI is performed using the micromanipulator and – injector setup, which enable to simply the cutting–edge workflow.

You have never ever imagined how smart and sophisticated devices are! An inverted microscope equipped with Modulation Contrast, two micromanipulators (one for moving the holding capillary and another for collecting and transferring the spermatozoa), adapter for an inverted microscope, air microinjector for holding the oocyte and oil microinjector for transferring the sperm – this is the “smart space” where the embryos are created.

ICSI microtools 

After that, embryologists prepare consumables and media embryo–tested light mineral oil, shallow cell culture dishes, holding micropipette for holding of oocytes, ICSI transfer injection micropipette, for collecting and injecting sperms, culture media (supplemented with antibiotics, protein, and pyruvate), PVP (polyvinylpyrrolidone) or equivalent formulations.

ICSI dish

ICSI needs some space to be performed. Therefore, embryologist prepares the microinjection dish. The first thing that it is done – is heating. It is essential to heat all media and the oil to 37 °C prior to use.

After that, several droplets of the medium are placed in the center of the cell culture dish. Tiniest droplets intended for retrieved spermatozoa are supplemented with PVP before the addition of the sample.

Additional droplets containing PVP only may be necessary for storage of selected spermatozoa before ICSI Microinjection. All droplets are completely covered with light mineral oil to maintain their stability, temperature, and pH. Once prepared, the microinjection dish can be placed into the incubator until use.

When the micromanipulator workstation is set up, the embryologist must confirm that there are no air bubbles in the system. If there are no bubbles in the system, the embryologist prepares the microinjection pipettes (capillaries).

Both micropipettes (ICSI Injection Micropipette and ICSI Holding Pipette) need to be accurately positioned into the holding system of the microinjectors, then aligned and equilibrated before the ICSI procedure is started.

As the ICSI holding micropipette has a greater diameter than the ICSI injection micropipette, an embryologist uses it as a guide for the positioning and equilibration steps.

First, the ICSI micropipettes are integrated into the micropipette holder, which is connected to the microinjector via a tube. For holding of oocytes, an air-based microinjector is often preferred. For the injection side, many embryologists prefer an oil-based microinjector because it allows highly precise control of the sperm–sample to be injected inside the oocyte.

ICSI procedure is physically stressful for the oocyte because it may be torn. That is why depth control of microinjection is vital for preventing the oocyte tearing.

The holding micropipette is usually aligned in the vertical position so that the micropipette holder can be rotated, even when the holding pipette is tightly gripped in place. In contrast, the ICSI injection micropipette tilts slightly downwards so that the tail of the spermatozoon can be broken properly.

The next step of embryologist is to prime ICSI injection micropipette and holding micropipette with ICSI media (medium) before use so that the sperm and oocytes never come into contact with air or oil.

Selection and “catching” of sperm for microinjection

For selection and “catching” of sperm for microinjection Micromanipulator for ICSI has the special tool – the Dual Speed joystick with fine & extra fine speed mode and with dynamic speed mode. These speed modes enable intuitive movement during injection in all three dimensions. The Dual Speed joystick allows controlling the speed and fluidity in everything embryologist does.

If the dynamic mode is activated, the needle proceeds in the desired direction showing the fastest performance. Dynamic speed mode is activated when embryologist is “catching” spermatozoa.

If the embryologist needs the highest accuracy in selection or needs more time, the needle can be moved carefully in the fine or extra fine speed mode whilst still capable of a considerable range of quick motion once the dynamic, outer zone of the joystick is entered. The same extra fine mode is activated when ICSI is performed.

ICSI Microinjection

Sperms have the tails! And their tails allow embryologists to catch them! Sounds amusing, doesn’t it? Want to catch that dude? Catch the tail first!

The necessary amount of the sperm sample is loaded into a drop pre–filled with PVP, whilst oocytes are placed into the designated medium drops. The embryologist presses joystick key twice to lower the ICSI transfer micropipette to the necessary position, and under 200x to 400 magnification a spermatozoon is selected and immobilized either by “rolling” the ICSI transfer micropipette over the tail or by gently pressing the tail against the bottom of the dish.

The spermatozoon is aspirated, tail–first into the ICSI transfer micropipette (ICSI injection micropipette) as delicately as possible. The joystick key is then pressed twice to move the transfer injectable micropipette containing the single top–quality spermatozoon up into the overlay medium. The cell culture dish is moved to one of the drops containing the single oocyte.

The embryologist presses the joystick to move the holding micropipette from one position to another position. The oocyte is attached gently but firmly to the holding micropipette by the negative pressure created by the Air microinjector for holding the oocyte.

Injection of the oocyte is normally undertaken with the first polar body being positioned at either 6 o’clock or 12 o’clock.

To achieve 6 o’clock or 12 o’clock orientation of the first polar body, it may be necessary to turn the oocyte. Oocyte turning can be done by the ICSI transfer micropipette which is lowered to the necessary position and slightly varying the negative pressure of the air microinjector for holding the oocyte until the polar body has reached the desired position.

After that, the ICSI injection micropipette is focused in the same plane as the maximal diameter of the oocyte becomes noticeable.

By rotating the knob of the Oil microinjector for transferring the sperm, the spermatozoon is gently moved along the capillary until it is positioned at the very tip.

By slight moving of the joystick, the embryologist pushes the transfer micropipette (ICSI Injection Micropipette) with utmost accuracy through the zona pellucida and through 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 delicately and accurately broken not torn. For preventing the oocyte tearing, a small amount of ooplasm is gently aspirated into the injection micropipette as a sign of membrane rupture. The aspirated ooplasm and the spermatozoon are then deposited towards the center of the oocyte.

For minimization the volume of medium and PVP introduced into the cytoplasm, the transfer micropipette is gently withdrawn after the head of the sperm cell has left the capillary tip. Once this has been completed, the injected oocyte is released from the holding capillary, and both capillaries are returned to the basic position by pressing the joystick key twice.

If several oocytes are to be injected with sperm, only 3 to 6 oocytes are placed in the cell culture dish at the same time to avoid stress to the oocytes (temperature and pH changes). Once the injection procedure is completed, the oocytes are placed into an appropriate culture medium and incubated overnight.

Timeline for fertilization and Embryo Transfer

Fertilization occurs approximately 15 to 18 hours after microinjection. Normally fertilized oocytes contain two pronuclei and two polar bodies. Embryo Transfer into the uterus is performed 2 to 5 days after ICSI.

Assisted Hatching (AH) what is that? 

The embryo appears from the fertilized oocyte. And we all know that. But how fertilized oocyte becomes an embryo? Everything is simple! When there are too many embryonic cells inside the oocyte, to turn into the embryo, the oocyte should be torn. Imagine a huge birthday balloon with tiny bundle–balloons inside. When there are too many smaller balloons are placed inside, the huge balloon bursts!

In the same way, the embryo does! The embryo should tear its envelope to implant successfully in the womb. To hatch “outside” its basic envelope called “zona pellucida.” The embryo must spontaneously rupture through zona pellucida layer, hatch outside and only after that the successful implantation occurs. If there is no hatching, the embryo dies.

That is why Assisted Hatching was designed for IVF. It is usually performed on day three (Day 3) after fertilization.

During Assisted Hatching, the zona pellucida, which envelopes the embryo is thinned or ruptured. The outer layer of the zona pellucida is dissolved with an acidic mixture. Alternately, a laser may be used to drill the tiny hole in the zona pellucida. But most often a Zona drilling micropipette is used to break open the outer layer of the zona pellucida.

The embryo will be transferred into uterus a day after Assisted Hatching.

If there are other complications that should be prevented, for example, the genetic content should be checked for monogenic diseases and/or numerical/structural chromosomal abnormalities, the PGD procedure is performed.

If PGD (Blastomere Biopsy and Polar Body Biopsy) should be performed, the biopsy micropipettes are prepared and embryo biopsy is performed.