How the ISCI procedure is performed – the treatment step by step

By (gynecologist), (gynecologist), (gynecologist), (clinical embryologist), (embryologist), (embryologist) and (biochemist).
Last Update: 09/06/2022

Intracytoplasmic sperm injection, abbreviated as ICSI, is one of the steps of in vitro fertilization (IVF) treatment. Specifically, ICSI refers to the method of fertilizing oocyte with sperm.

However, many people often use the term ICSI or IVF-ICSI to refer to the entire fertility treatment process. Therefore, in this article we are going to detail step by step all the phases of IVF-ICSI.

What does treatment with ICSI involve?

ICSI has procedures that are very similar to the parts of the conventional IVF process. The difference lies in the way fertilization occurs once oocytes and sperm have been obtained in the laboratory.

The ICSI process step by step is as follows:

Ovarian Stimulation

Ovarian stimulation consists of the administration of hormonal medication in order to make several ovarian follicles mature at the same time. This makes it possible to obtain a greater number of egss to fertilize and increase the possibility of pregnancy.

In addition, ovarian stimulation controls the woman's menstrual cycle and prevents spontaneous ovulation from taking place, which would lead to the failure of the IVF-ICSI cycle.

Before starting ovarian stimulation, it is usual to prescribe the contraceptive pill to patients in order to synchronize the menstrual cycle.

Depending on the duration of ovarian stimulation, there are two basic protocols in IVF patients:

  • Long protocol: starts before menstruation with injections of GnRH agonists, which are used to pause the pituitary gland and prevent the secretion of endogenous hormones. Once the period arrives, the previous injections are combined with those of gonadotropins to initiate multiple follicular development.
  • Short protocol: the number of injections is reduced, as stimulation with gonadotropins begins after the onset of menstruation. On day 8 of the cycle, injections with GnRH antagonists are administered to halt the the pituitary gland.

In recent years there has been a trend towards simplification of ovarian stimulation protocols thanks to the multitude of advantages it offers.

As Dr. Gorka Barrenetxea, a specialist in Gynaecology and Obstetrics, says:

For 10-15 years now, we have been using other adjuvant drugs, GnRH antagonists, in such a way that now the stimulation process lasts approximately 10 days.

Simplyfying the process means shortening the duration of the ovarian stimulation, and reducing the number of hormone injections.

The gonadotropin (FSH) administration phase usually lasts approximately 6-10 days. During this time, the patient will have to go to ultrasound control 2 or 3 times to check follicular growth.

You can read more about this step of the process in the following link: Ovarian Stimulation Protocols for IVF – Process & Medications Used.

Follicular Puncture

Also known as egg retrieval or ovum pickup the next step is follicular puncture, which consists of removing the mature oocytes with ultrasound guidance.

This is a simple 30-minute operation performed in the surgery room under anaesthetic to avoid discomfort to the patient.

About 30-34 hours before the ovarian puncture, the patient must have an injection of hCG hormone to trigger ovulation and induce the final maturation of the oocytes.

The embryologist Maria De Las Heras points out that:

Follicular puncture is not painful, as the patient is sedated when she enters the operating room.

Once in the laboratory, the follicular fluid must be examined in detail under microscope to locate the oocytes and transfer them to a plate with culture medium.

If you are interested in learning more about this phase of treatment, you can continue your reading in the next post: Ovum Pick-Up Procedure – How Are Eggs Harvested in IVF?

Oocyte Denudation

Oocyte denudation is the removal of the cell layer of granulose that surrounds the mature oocyte. To do this, it is necessary to wait a few hours after follicular puncture because the egg undergoes a final maturation during this time at rest.

There are two techniques for denuding oocytes:

  • Chemical Denudation: a medium is used with hyaluronidase, an enzyme that degrades the hyaluronic acid that binds the granulose cells together. In natural fertilization, hyaluronidase is secreted by the spematozoon in order to penetrate the egg.
  • Mechanical Denudation: the ovum is passed through pipettes of different calibre, from larger to smaller diameter, until all the surrounding cells are completely detached.

Usually, IVF laboratory protocols combine both methods to more effectively denudate eggs.

After denudation, it is necessary to check whether the oocytes are mature in order to perform ICSI. To do this, it is necessary to visualize the polar body in the perivitelline space of the ovum.

Oocyte denudation is a necessary step before intracytoplasmic sperm microinjection.

Sperm Collection & Preparation

While the women is undergoing follicular puncture, the man must leave a semen sample in the laboratory collected by masturbation, unless frozen or donor semen is to be used.

For the semen sample preparation, sperm capacitation is necessary: the seminal plasma is removed and the higher quality sperm are concentrated in a new culture medium.

There are several techniques for capacitating semen. You can find out how they are done in detail in the following article: What Is Capacitation of Sperm Cells? – Definition & In Vitro Techniques.

The objective is to obtain a sample of progressive motile sperm count (MSC) of approximately one million per milliliter, since if there were more spermatozoa it would be difficult to select them under the microscope.

On the other hand, in cases of severe male factor infertility, fewer and poorer quality sperm may be obtained. In fact, it is sometimes necessary to obtain the sperm with sperm asipiration or testicular biopsy to do ICSI.

However, although it is more complicated, ICSI can also be done under these conditions, as we only need the same number of live sperm as the number of oocytes are going to be injected.

Oocyte Fertilization

The way the ovum is fertilized is what differentiates the ICSI technique from conventional IVF.

As its name suggests, ICSI involves injecting the sperm directly into the cytoplasm of the egg. To do this, the following steps are carefully followed:

  • Initial Preparation: the holding and microinjection pipettes (ICSI) are placed under an inverted microscope. The ICSI plate is then prepared with drops of culture medium where the oocytes are placed on one side and the sperm on the other.
  • Sperm Selection: The sperm sample is studied, looking for the best quality and, once selected, must be immobilized with a quick movement of the ICSI pipette to fracture its tail. The sperm is then aspirated with this same pipette.
  • Oocyte Orientation: in order not to damage the internal structures of the ovum with the injection, it is placed with its polar body at the top and is held by the holding pipette so that it does not move.
  • Intracytoplasmic Injection: the ovum is gently pressed with the injection pipette through the zona pellucida and the internal membrane. Once inside the egg, a small amount of cytoplasm is aspirated to come into contact with the sperm, which is then gently introduced into the ovum.
  • Final evaluation: the ovum breakage type provides information about the oocyte quality and can condition its further development. Therefore, it is necessary to evaluate the type of breakage, which can be by pressure or aspiration.

Once the entire ICSI process has been completed, it is also important to note the morphological characteristics of the oocytes: polar body, cytoplasm, perivitelline space, zona pellucida, etc.

Finally, the oocytes are stored in culture plates in the incubator, waiting to assess whether fertilization has taken place. This takes approximately 24 hours.

Embryo culture

After fertilization and throughout embryonic development, it is necessary to evaluate the morphogenetic characteristics of the embryos in order to transfer those of better quality and with a greater possibility of implantation.

Depending on the stage of embryo development, some parameters will be evaluated:

  • Zygotes: approximately 18 hours post-fertilization are valued to see if the two polar bodies and the two pronuclei have appeared, which will indicate that fertilization has been successful.
  • 2-3 day embryos: the embryo has already divided and, therefore, the number of cells, their symmetry, fragmentation, multinucleation, if there are vacuoles, etc. are assessed.
  • 5-6 day blastocysts: at this stage, the embryo is already constituted by a multitude of cells that form the internal cell mass and the trophectoderm. The degree of expansion of the blastocysts and whether they have begun to leave the zona pellucida is also assessed, which is known as hatching.

For more information on the conditions and characteristics of embryo culture, you can read on in the next post: Embryo Culture for Human IVF.

Embryo Transfer

Depending on the characteristics of the treatment and the number of embryos obtained, the embryo transfer can be carried out on day 3 or day 5.

To do this, the best quality embryo or embryos will be selected on the basis of the previously mentioned parameters. You can find out how this embryo selection is done in the following link: Embryo Quality & Grading – Does It Affect IVF Success?

Prior to embryo transfer, the woman must have received estrogen and progesterone for optimal endometrial preparation.

The transfer procedure is very simple and does not require anesthesia. First, the selected embryo is taken with a thin catheter and then inserted through the woman's vagina into the uterus.

The embryo transfer procedure is very simple and does not require anesthesia. First, the selected embryo is taken with a fine catheter and then introduced through the woman's vagina into the uterus. Thanks to simultaneous ultrasound, it is possible to see how the embryo is left in the uterine cavity for implantation to take place.

In the following article you will find all the detailed steps of embryo transfer after an IVF treatment: IVF Embryo Transfer Procedure – Definition, Process & Tips.

Embryo Vitrification

In most IVF cycles, surplus embryos will remain after embryo transfer. To give an example, British legislation only allows the transfer of a maximum of 3 embryos. However, there is a growing trend towards the transfer of a single embryo instead of two or three. The main reason is to avoid the possibility of multiple pregnancies and the risks it may entail, both for the mother and the fetuses.

As a result, embryos that are not transferred at the first attempt are vitrified for future use, either because the first transfer has not been successful, or to be able to have a second child in the future. In order to vitrify the embryos, they must be of good or medium quality so that the survival of the embryos after devitrification can be guaranteed.

Another option for the remaining embryos would be to donate them to other patients or to research.

If you want to know what the freezing protocols are like today, don't miss the next post: What Is Embryo Vitrification? – Advantages over Slow Freezing.

Pregnancy test after ICSI

The patient will have to wait about 9-12 days to do a pregnancy test and check if the IVF-ICSI has been successful. This test can be performed on either urine or blood and the amount of the beta hormone hCG is measured.

For this reason, this period between the embryo transfer and the confirmation of the pregnancy is commonly known as the two-week-wait. Specialist recommend continuing with daily life and avoiding thinking about whether the treatment will have been successful during the entire period.

If you need to undergo IVF to become a mother, we recommend that you generate your Fertility Report now. In 3 simple steps, it will show you a list of clinics that fit your preferences and meet our strict quality criteria. Moreover, you will receive a report via email with useful tips to visit a fertility clinic for the first time.

FAQs from users

What is ICSI?

By Dr. José León Tovar M.D., M.Sc. (gynecologist).

The big difference between conventional IVF and ICSI is that in ICSI each egg is microinjected with a sperm. So the fertilization, let's say, is more controlled. It is not a deposit of eggs with a deposit of sperm, but a sperm is introduced directly into each egg and fertilization evaluated after 24 hours.

By José María Sánchez Jordán M.D. (gynecologist).

There is no medical contraindication for a repeat ICSI cycle, therefore, no maximum number of attempts has been established. I would recommend a total of three attempts if there has been no success during previous cycles.

Is it possible to do an intracytoplasmic injection with slow sperm?

By Zaira Salvador B.Sc., M.Sc. (embryologist).

Yes, the most important thing to do for an ICSI is to make sure that the sperm are alive. In asthenozoospermic semen samples, with reduced sperm motility, we will try to select the sperm that can move as straight as possible and have good morphology.

When is PGD done after ICSI?

By Zaira Salvador B.Sc., M.Sc. (embryologist).

The preimplantation genetic diagnosis or PGD consists of the genetic analysis of an embryo cell to know if it suffers any genetic alteration. This cell biopsy is normally done 3 days after ICSI when the embryo has approximately 8 cells. However, it is also possible to do PGD with blastocysts, by biopsying several cells of the trophectoderm.

For more information related to this topic, you can follow the reading by clicking here: What Is PGD or Preimplantation Genetic Diagnosis?

When is it possible to do a pregnancy test after ICSI?

By Zaira Salvador B.Sc., M.Sc. (embryologist).

In both IVF and ICSI, it is recommended to do a pregnancy test at least 14 days after the follicular puncture. Thus, the risk of false positive or false negative is minimal. However, it is best to do the beta-hCG analysis before a urine test to know if it has been successful, as the beta test is more reliable.

If you are interested in ISCI and need more information about it, we recommend reading this article: Intracytoplasmic sperm injection: What is ICSI and how much is it?

We have talked about the current tendency of assisted reproduction clinics using simplified ovarian stimulation protocols. If you would like to read about this in more detail please visit the following post: What is a short IVF protocol with GnRH antagonists?

The embryo transfer can take place of day 3 or day 5 of embryo culture. Both options carry a series of advantages and disadvantages that should be evaulated for each individual case. To find out more about this topic you can continue your reading in the following article: When to do the embryo transfer?

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Zulategui, J; Cobo, A; Romero, J; Galán, A; Albert, C. y De los Santos, M.J. Inyección intracitoplasmática de espermatozoides (ICSI). En: Remohí, J; Cobo, A; Romero, J; De los Santos, M.J. y Pellicer, A. (2008). Manual práctico de esterilidad y reproducción humana. Madrid: McGraw-Hill.

Palermo, G; Cohen, J; Alikani, M; Adler, A. and Rosenwaks, Z. (1995). Development and implementation of intracytoplasmic sperm injection (ICSI), Reprod Fertil Dev, 7(2):211-7.

Vidéo ReproduccionAsistida.Org: Cómo son los tratamientos de estimulación ovárica, by MD Gorka Barrenetxea, on October 6/2017 [See original video in Spanish here]

Vidéo ReproduccionAsistida.Org: ¿es doloroso la punción folicular?, by Embryologist María De Las Heras, on July 19/2016 - [See original video in Spanish here]

FAQs from users: 'What is ICSI?', 'How many ICSI attempts are recommended?', 'Is it possible to do an intracytoplasmic injection with slow sperm?', 'When is PGD done after ICSI?' and 'When is it possible to do a pregnancy test after ICSI?'.

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Authors and contributors

 Gorka Barrenetxea Ziarrusta
Gorka Barrenetxea Ziarrusta
M.D., Ph.D.
Bachelor's Degree in Medicine & Surgery from the University of Navarra, with specialty in Obstetrics and Gynecology from the University of the Basque Country. He has over 30 years of experience in the field and works as a Titular Professor at the University of the Basque Country and the Master's Degree in Human Reproduction of the Complutense University of Madrid. Vice-president of the SEF. More information about Gorka Barrenetxea Ziarrusta
License: 484806591
Dr.  José León Tovar
Dr. José León Tovar
M.D., M.Sc.
José León Tovar has studied medicine and has specialized in the field of assisted human reproduction. Currently, Dr. Tovar is the head of the Assisted Reproduction Unit at the Ginemed Huelva center. More information about Dr. José León Tovar
Member number: 414115772
 José María  Sánchez Jordán
José María Sánchez Jordán
Dr. José María Sánchez has a degree in Medicine and Surgery from the Faculty of Medicine of Malaga and specialized in Obstetrics and Gynecology. More information about José María Sánchez Jordán
Member number: 511104002
 María de Las Heras Martínez
María de Las Heras Martínez
B.Sc., M.Sc.
Clinical Embryologist
Bachelor's Degree in Biology from the Pompeu Fabra University and Master's Degree in Biology of Reproduction & Assisted Reproductive Technologies from the Autonomous University of Barcelona, in collaboration with Instituto Universitario Dexeus. Master's Degree in Biochemical Research from the Basque Country University. Clinical Embryologist by the ESHRE. More information about María de Las Heras Martínez
 Marta Barranquero Gómez
Marta Barranquero Gómez
B.Sc., M.Sc.
Graduated in Biochemistry and Biomedical Sciences by the University of Valencia (UV) and specialized in Assisted Reproduction by the University of Alcalá de Henares (UAH) in collaboration with Ginefiv and in Clinical Genetics by the University of Alcalá de Henares (UAH). More information about Marta Barranquero Gómez
License: 3316-CV
 Zaira Salvador
Zaira Salvador
B.Sc., M.Sc.
Bachelor's Degree in Biotechnology from the Technical University of Valencia (UPV). Biotechnology Degree from the National University of Ireland en Galway (NUIG) and embryologist specializing in Assisted Reproduction, with a Master's Degree in Biotechnology of Human Reproduction from the University of Valencia (UV) and the Valencian Infertility Institute (IVI) More information about Zaira Salvador
License: 3185-CV
Adapted into english by:
 Michelle Lorraine Embleton
Michelle Lorraine Embleton
B.Sc. Ph.D.
PhD in Biochemistry, University of Bristol, UK, specialising in DNA : protein intereactions. BSc honours degree in Molecular Biology, Univerisity of Bristol. Translation and editing of scientific and medical literature.
More information about Michelle Lorraine Embleton

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