Intracytoplasmic sperm injection (ICSI)

 

Intracytoplasmic sperm injection (ICSI) is an in vitro fertilization procedure in which a single sperm is injected directly into an egg. This procedure is most commonly used to overcome male infertility problems, although it may also be used where eggs cannot easily be penetrated by sperm, and occasionally as a method of in vitro fertilization, especially that associated with sperm donation. The ICSI technique was developed by Gianpiero Palermo around 1991 at the Vrije Universiteit Brussel, in the Center for Reproductive Medicine headed by Paul Devroey and Andre Van Steirteghem.
ICSI is a very effective method to get fertilization of eggs in the IVF lab after they have been retrieved from the female partner. IVF with ICSI involves the use of specialized micromanipulation tools and equipment and inverted microscopes that enable embryologists to select and then pick up individual sperms in a tiny specially designed hollow ICSI needle. Then the needle is carefully advanced through the outer shell of the egg and egg membrane and the sperm is then injected into the inner part (cytoplasm) of the egg. This will usually result in normal fertilization in approximately 70-85% of eggs injected with viable sperm. First, the woman must be stimulated with medications and have an egg retrieval procedure so that we can obtain several eggs in order to attempt in vitro fertilization and ICSI.

The ICSI procedure is done under a microscope using multiple micromanipulation devices (micromanipulator, microinjectors and micropipettes). A holding pipette stabilizes the mature oocyte with gentle suction applied by a microinjector. From the opposite side a thin, hollow glass micropipette is used to collect a single sperm, having immobilized it by cutting its tail with the point of the micropipette. The micropipette is pierced through the oolemma and into the inner part of the oocyte (cytoplasm). The sperm is then released into the oocyte. After the ICSI procedure, the oocyte will be placed into cell culture and checked on the following day for signs of fertilization.

In natural fertilization sperm compete and when the first sperm penetrates the oolemma, the oolemma hardens to block the entry of any other sperm. Concern has been raised that in ICSI this sperm selection process is bypassed and the sperm is selected by the embryologist without any specific testing. However, in mid 2006 the FDA cleared a device that allows embryologists to select mature sperm for ICSI based on sperm binding to hyaluronan, the main constituent of the gel layer (cumulus oophorus) surrounding the oocyte. The device provides microscopic droplets of hyaluronan hydrogel attached to the culture dish. The embryologist places the prepared sperm on the microdot, selects and captures sperm that bind to the dot. Basic research on the maturation of sperm shows that hyaluronan-binding sperm are more mature and show fewer DNA strand breaks and significantly lower levels of aneuploidy than the sperm population from which they were selected. A brand name for one such sperm selection device is PICSI.

There is some suggestion that birth defects are increased with the use of IVF in general and ICSI specifically, though results of different studies differ. In a summary position paper, the Practice Committee of the American Society of Reproductive Medicine has said it considers ICSI safe and effective therapy for male factor infertility, but may carry an increased risk for the transmission of selected genetic abnormalities to offspring, either through the procedure itself or through the increased inherent risk of such abnormalities in parents undergoing the procedure.

Who should be treated with intracytoplasmic sperm injection?

There is no "standard of care" in this field of medicine regarding which cases should have the ICSI procedure and which should not. Some clinics use it only for severe male factor infertility, and some use it on every case. The large majority of IVF clinics are somewhere in the middle of these 2 extremes. Our thinking has changed over time - we are now doing more ICSI (as a percentage of total cases) than in the past. Certainly, as we learn more about ways that we can help couples conceive, our thinking in this area will continue to evolve. Having said that, we are currently recommending in vitro fertilization (IVF) with ICSI for:

  • All couples with severe male factor infertility that do not want donor sperm insemination.
  • All couples with infertility with: 
    • Sperm concentrations of less than 15-20 million per milliliter
    • Sperm motility less than 35%
    • Very poor sperm morphology (subjective - specific cutoff value is not appropriate)
  • All couples having IVF who have had a previous cycle with no fertilization - or a low rate of fertilization (low percentage of mature eggs that are normally fertilized).
  • All couples having IVF who have a very low yield of eggs at the egg retrieval - our current cutoff is 5-6 (or less) eggs. In this scenario, ICSI is being used to try to get a higher percentage of eggs fertilized than with conventional insemination of the eggs (just mixing eggs and sperm together). 


How is ICSI performed?

ICSI is typically used in cases of severe male infertility, including: 

  • Very low sperm count (also known as oligospermia) 
  • Abnormally shaped sperm (also known as teratozoospermia) 
  • Poor sperm movement (also known as asthenozoospermia) 

If a man does not have any sperm in his ejaculate, but he is producing sperm, they may be retrieved through testicular sperm extraction, or TESE. Sperm retrieved through TESE require the use of ICSI. ICSI is also used in cases of retrograde ejaculation, if the sperm are retrieved from the man’s urine. ICSI may also be done if regular IVF treatment cycles have not achieved fertilization. 

ICSI is done in a superovulated cycle during which fertility drugs (human menopausal gonadotropin - HMG- injections) are administered to the wife to aid in the production of multiple eggs, which are then removed under vaginal ultrasound guidance as is done for IVF. 
In normal circumstances, the egg is surrounded by a cluster of cells known as the cumulus corona cells, and this is called the oocyte cumulus corona complex. 

These cumulus cells are removed by repeated passage of the oocyte cumulus corona complex through fine pipettes, and by treating them with a chemical called hyaluronidase so that these cells are stripped off. The denuded eggs are examined, and only mature eggs (eggs in metaphase II, which have a polar body) are used for ICSI.

Sperm is collected from the man, usually through masturbation. For men with severe oligospermia, we have found it useful to use sequential ejaculates. Even though the first semen sample may not contain any sperm, we often find motile sperm in the second (or even the third sample, for men with enough stamina!). This maybe because the later samples contain "fresher" sperm. Since these samples contain such few sperm, they need to processed very carefully, so that the all the sperm in the sample are recovered in the culture medium, and can be used for ICSI.

For men with variable sperm counts, which vary from zero to a few thousand, it may be helpful to freeze a sample (which contains sperm) in advance. For patients with azoospermia, sperm harvesting techniques need to be used to retrieve the sperm. For men with obstructive azoopsermia, (because of duct blockage or absence of the vas deferens), the simplest technique is called PESA (percutaneous epididymal sperm aspiration), in which the sperm is sucked out from the epididymis by puncturing it with a fine needle. Occasionally, one may have to use microsurgery to find epididymal sperm, and this is called MESA (microepididymal sperm aspiration). 

Fertilization and pregnancy success rates with ICSI

Fertilization rates for ICSI: Most IVF programs see that about 70-85% of eggs injected using ICSI become fertilized. We call this the fertilization rate, which is different from the pregnancy rate.

Pregnancy success rates for in vitro fertilization procedures with ICSI have been shown in some studies to be higher than for IVF without ICSI. This is because in many of the cases needing ICSI the female is relatively young and fertile (good egg quantity and quality) as compared to some of the women having IVF for reasons other than male factor infertility. Another way to say this is - average egg quantity and quality is usually better in ICSI cases (male factor cases) because it is less likely that there is a problem with the eggs - as compared to cases with unexplained infertility in which there is more probability of a somewhat reduced egg quantity and quality (on the average, since some women in this group have egg related issues).
ICSI success rates vary according to the specifics of the individual case, the ICSI technique used, the skill of the individual performing the procedure, the overall quality of the laboratory, the quality of the eggs, and the embryo transfer skills of the infertility specialist physician performing the procedure. 

Sometimes IVF with ICSI is done for "egg factor" cases - low ovarian reserve situations. This is when there is either a low number of eggs, or lower "quality" eggs (or often both). In such cases, ICSI fertilization and pregnancy success rates are somewhat lower (as a group) since the main determinant of IVF success is the quality of the transferred embryos - and the quality of the eggs is the most crucial factor determining the quality and viability of the resulting embryo.

In some cases, assisted hatching might be done on the embryos prior to transfer, in order to maximize chances for pregnancy.
IVF success depends on a certain number of spermatozoa present in the ejaculated, having good motility and morphology. Riedel et al. the minimum andrological requirements for a conventional IVF; 5 x 106/ml concentration, 30% progressive motility and 30% normal morphology. Men having parameters lower than the appointed values had bad prognostics. Nowadays, however, the best approach to this kind of male infertility is ICSI: only one living spermatozoon is needed for each M II oocyte (oocyte in the metaphase II cell stage – that means – mature).
Before the ICSI era, various attempts were made to modify and refine conventional IVF in order to enhance pregnancy rates in cases of male infertility. The use of high sperm concentration showed good results in oligospermy and oligoasthenospermy. However, sperm morphology plays an important role in the success of this type of IVF. 

More recently, when ICSI was already available, comparative studies have been made between high sperm concentration IVF and ICSI, in the cases of severe teratozoospermia. Higher fertilization rates and better embryo quality were obtained through ICSI when compared to high sperm concentration IVF. A clear tendency for better pregnancy and implantation rates was observed in the ICSI group. Nowadays ICSI clearly surpassed of modified conventional IVF techniques, including high sperm concentration insemination, for the treatment of severe male factor infertility.

ICSI is not strictly indicated for morphological alterations of spermatozoa, but also for low sperm motility and concentration. A summary of four years of ICSI practice indicated that similar results are obtained by ICSI using abnormal semen and conventional IVF using normal semen.

ICSI with ejaculated sperm can be successfully used in patients with fertilization failure after conventional IVF and also in patients who cannot be accepted for these procedures due to an ejaculated having less than 500.000 progressive mobile spermatozoa. High fertilization and pregnancy rates can be obtained when a mobile spermatozoon is injected. The injection of an immobile or dead spermatozoon results in low fertilization rates. Absolute asthenospermia is a sporadic condition except for sperm ultra-structural defects. Therefore, if immobile sperm injection in an initial cycle leads to bad results, subsequent ICSI cycles are justified. 

In case we find only immobile spermatozoa for microinjection, it is important to select the living ones, and this can be achieved by using the hypo-osmotic swelling test (to look for a swelling in the spermatozoon tail): living spermatozoa having an intact cell membrane show a typical swelling when placed inside a hypo-osmotic saline solution. Only these spermatozoa must be selected for the ICSI procedure. Of course the hypo-osmotic test must be preceded by the eosine Y coloration, to exclude complete necrozoospermia. When only dead spermatic cells are present in the ejaculated, using testicular sperm is indicated. Other seminal parameters, like concentration, morphology (except for globozoospermia) and high anti-sperm antibody rates do not influence ICSI success rates. Success with ICSI has been described for patients with acrosome absence. 

Any infertility form associated to excretion duct obstruction can be treated by ICSI, by microsurgically removing sperm from the epidydim or testicle. Obstructive azoopsermia can result from congenital bilateral absence of vas deferens (CBAVD), vasectomy or vasoepidydimostomia reversion failure. Epidydim sperm is obtained mainly by microsurgery of epidydim and sperm aspiration (MESA), under general anesthesia. Other used method is percutaneous epididym sperm aspiration (PESA) preceded by local anesthesia. When immobile sperm is removed from the epidydim, due to an epididymary fibrosis, we can try to remove sperm from the testicles through a biopsy. Two techniques are used to obtain testicular tissue: A biopsy or a thin needle aspiration. Testicular biopsy has also been used in some non obstructive azoopsermia cases. In patients having severe testicular deteriorization and germ cell aplasia (Sertoly cell syndrome only), hypospermatogenesis or incomplete maturation, sometimes sperm can be found after multiple biopsies. Sperm removal may not always be successful in all azoospermic patients. However, there is no precise indicator for testicular sperm removal except testicular histopathology. Optimal spermatozoa removed by testicular biopsy can be obtained by a delicate tissue wash. Very frequently can we find living spermatozoa after enzymatic treatment of testicular samples, for red cell removal.

Cryopreservation of supranumerary spermatozoa removed from the epidydim or testicle has na important function, because frozen or unfrozen sperm microinjection may be used, avoiding surgery in future ICSI cycles. Successful ICSI using frozen epididymal sperm has been described. This also holds true for pregnancy resulting therefrom. Testicular sperm cryopreservation is less frequently used and more difficult, because only a limited number of spermatozoa is present for good results. Cohen et al. have recently described a cryopreservation method with reduced spermatozoon number inside the empty oocyte zona pellucida; although adapting this method for testicular biopsy may take excessive time.

It must be added that ICSI method may not be used in approximately 3% of the cycles. This may result from the fact that the cumulus-corona complex or Metaphase II oocytes are not available, or there couldn’t be found any sperm in testicular biopsies in patients with non obstructive azoospermia.
Due to excellent fertilization rates and good embryo development, ICSI has been indicated for practically all infertility cases, even in the absence of a male infertility factor.

How can ICSI be used to treat men with a zero sperm count (azoospermia)? 

For patients with obstructive azoopsermia in whom sperm cannot be found in the epididymis, it is always possible to find sperm in the testis. The easiest way to retrieve this is through TESA or testicular sperm aspiration, in which the testicular tissue is sucked out through a fine needle, under local anaesthesia. The testicular tissue is placed in culture media and sent to the lab, where it is processed. The sperm are liberated from within the seminiferous tubules (where they are produced) and are then dissected free from the surrounding testicular tissue.

Using sperm from the epididymis and testis for ICSI in order to treat patients with obstructive azoospermia is logical, and thus conceptually easy to understand. However, surprisingly, it is possible to find sperm even in patients who have testicular failure (nonobstructive azoospermia) - even in those men with very small testes. The reason for this is that defects in sperm production are "patchy"- they do not affect the entire testis uniformly. 
This means that even if sperm production is absent in a certain area, there may be other areas in the testis where sperm production would be normal (this could be because the genetic defect that causes abnormal spermatogenesis may be "leaky"). Since such few sperm are needed for ICSI, we can find enough sperm in over 50 per cent of patients with testicular failure, even if their testes are as small as a peanut!

What is TESE (testicular sperm extraction) ICSI? 

However, while finding sperm is quite easy in men with obstructive azoospermia (since their testes are functioning normally), patients with nonobstructive azoospermia (testicular failure) can be very challenging. Often, sperm production in these men is sparse, and multiple sites in the testis may need to be sampled before being able to find sperm. This can be done by performing mutiple tiny microbiopsies, and this is called TESE or testicular sperm extraction. (One of our patients suggested that we call this procedure TSEICSI - which stands for testicular sperm extraction with ICSI, and pronounce it as "sexy"!) This can be done through the needle, or as an open procedure performed under direct vision through a tiny skin incision under local anesthesia and sedation. 

Finding sperm in the testicular tissue can be a laborious process, depending on the degree of sperm production, and for some men with partial testicular failure, it can take upto 2-3 hours to find the sperm. Also, testicular sperm are technically hard to work with in the laboratory and only some IVF clinics have the requisite expertise. For men with nonobstructive azoopsermia, some clinics perform the TESE the day prior to egg retrieval, because they believe culturing the testicular tissue in the incubator for 24 hours helps the sperm to acquire motility, which makes them easier to work with. In case no sperm are found, either the couple decides to cancel the egg retrieval and abandon the cycle, or to go ahead with using donor sperm for IVF, as a backup option.

In patients in whom surgery needs to be performed in order to recover testicular or epididymal sperm, it is now possible to freeze the excess sperm. These sperm can then be thawed and used in future cycles in needed, thus sparing the patient the need for repeated surgery for sperm retrieval.
Once eggs and sperm have been collected, the actual process of injecting a single sperm into the egg is carried out in a laboratory. The injection is performed on a heating stage, on a specialized inverted microscope (which allows one to magnify details up to 400 times) equipped with Hoffman modulation contrast optics (which enhance "optical contrast", so that the details of the egg can be visualized easily). The precise control that is needed for microinjection is provided by using specialized micromanipulators, which allow one to execute very fine movements.

The eggs and sperm are manipulated using fine glass pipettes, made of thin capillary tubing, which are even finer than a human hair. These are custom made, the holding pipette being designed to hold a single sperm. Live sperm are placed in a drop of viscous polyvinyl pyrrolidone (PVP) solution, which serves to slow down the activity of the sperm. (It is helpful to slow down the sperm, so that they can be picked up more easily by the injecting needle.) A single sperm is then selected and its tail is pinched or broken to immobilize it. This is usually done by crushing the sperm tail by rolling it between the injection pipette and the base of the petri dish. It is essential to immobilize the sperm, so that it cannot move after it has been injected into the egg. A single immobile sperm is then picked up by sucking it into the injection pipette.

The egg is secured in place by applying gentle suction to its shell (the zona) with a holding pipette. The sperm is then injected directly into the centre (cytoplasm) of the egg by moving the injection pipette very precisely with the help by moving the injection pipette very precisely with the help of the micromanipulator into the egg, and then blowing the sperm out very gently into the cytoplasm of the egg. In order to do this, it is important to breach the zona of the egg and the outer membrane of the egg. The skill of the embryologist is a critical factor in the success of the ICSI process. After injecting the sperm, the pipette is withdrawn. Remarkably, once the injecting pipette is withdrawn, the egg will close and assume its original shape within 60 seconds. One can visualize ICSI as the sperm being given a "piggyback" ride into the egg, so that what the sperm cannot accomplish on its own, the laboratory does for it! The only requirement for ICSI is that the sperm should be alive, and there should be as many sperm as there are eggs.

Once all the eggs are injected with a single sperm each, they are placed in the CO2 incubator, and then observed approximately 14 hours later to see if fertilization has taken place. If fertilization has occurred, the 2-4 cell embryos can be transferred into the wife’s uterus about 48-72 hours after ICSI, as is done for IVF. Interestingly, embryo implantation rates in these patients are quite high, because the wives are usually young and completely normal.

Fertilization rates in the range of 60-80 per cent have been achieved in experienced hands-which means, of 100 microinjection eggs about 60 form embryos after ICSI. In fact the technology is now reliable enough to virtually guarantee fertilization, if there are sufficient good quality eggs. The pregnancy rate in one ICSI cycle is about 35 percent. Remarkably, the chance of achieving a pregnancy does not depend upon the sperm count or number (since you only need as many sperm as there are eggs!), but rather on the number and quality of eggs retrieved, which, in turn, depend upon the woman’s age. The risk of having a baby with a birth defect is not increased with this technique.

What are the risks of doing ICSI? 

More than 100,000 babies have been born worldwide after ICSI, and detailed studies have shown that there is no increased risk of birth defects or genetic anomalies, as a result of the technique.

It is possible, however, that some of the male children born as a result of this technique may be infertile as well (for example, if the cause for the testicular failure is a defective genetic locus, such as a microdeletion on the Y chromosome).