Microinjection: The Latest Advance in Treating the Infertile Man

The introduction of Microinjection Technology into the in vitro fertilization laboratory has revolutionized our treatment of the infertile man. Intracytoplasmic sperm injection, or ICSI, is a new infertility treatment that uses micromanipulation technology for treating male infertility. What ICSI promises is the possibility for every man to father his own baby --- no matter what his medical problem!

What exactly is ICSI? As the name suggests, ICSI is a technique in which a single sperm is injected into the center of the cytoplasm of the egg, in order to achieve fertilization. While this may sound very crude, ICSI allows the IVF laboratory to achieve fertilization with very few sperm. The beauty of the technique is that since the sperm is being injected directly into the egg, all that is needed to achieve fertilization are live sperm - no matter how abnormal these may appear to be. With ICSI the equation "1 egg plus 1 sperm = 1 embryo" becomes possible!

The Procedure for ICSI

ICSI is done in a super ovulated 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 may be because the later samples contain "fresher" sperm. Since these samples contain such few sperm, they need to be processed very carefully, so that 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). 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 anesthesia. 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 could 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!

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. Performing multiple tiny micro biopsies can do this, 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 testicular sperm are hard to work with in the laboratory. For men with nonobstructive azoopsermia, we usually perform the TESE the day prior to egg retrieval, because 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 if 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). Using specialized micromanipulators, which allow one to execute very fine movements, provides the precise control that is needed for microinjection.

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 injection 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. Sucking it into the injection pipette then picks up a single immobile sperm.

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 center (cytoplasm) of the egg 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 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 embryologist 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 two to four 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 microinjected 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.

ICSI is very expensive at present, because of the advanced technology it utilizes. Nevertheless, it is now available in most of India's large cities, and as times goes by, it is hoped that the cost of this procedure will decrease, making it affordable for more patients.

Some IVF clinics have started performing ICSI routinely for all patients, instead of offering them IVF. However, this is inappropriate. ICSI should be reserved for only two groups of patients: (1) those who have severe male factor infertility, for whom IVF is not a treatment option; and (2) those patients whose sperm have not been able to fertilize the eggs in an IVF cycle (total fertilization failure). Remember that pregnancy rates with ICSI are no better than with IVF, as long as fertilization occurs.

The Risk Factor

ICSI is still a new technique, and even though more than 10,000 babies have been born worldwide after it has come into use, and detailed studies have shown that there is no increased risk of birth defects after ICSI, nevertheless, it should only be reserved for those patients for whom traditional IVF is not a valid treatment option.

It is also possible 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 micro deletion on the Y chromosome).

Recent Advances

However, for some patients with severe testicular failure, sometimes it is not possible to find any sperm at all even in spite of taking multiple testicular biopsies. In such patients pregnancies have been achieved even by injecting round spermatids (immature precursor cells from which the sperm are formed) from the testis into the egg. This is now an area of intense research all over the world.

For men with no testes at all, the only technologic solution today would be cloning using nuclear transfer technology. This involves inserting the nucleus from an ordinary cell of the man (which contains all his DNA) into his wife's enucleated unfertilized egg (the nucleus of which has been removed) and then activating it by electro fusion. While cloning has been performed successfully in many animal species, it has never been used for treating humans so far.

Credits: Dr. Aniruddha Malpani, MD and DR. Anjali Malpani,