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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 ( ICSI Video ) 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 After injecting the sperm, the pipette is withdrawn. Remarkably, once the injecting pipette is withdrawn, the egg will close and assume its original shape within 60 seconds.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.
Fig 1. A view of the micromanipulator
Fig 2. A single sperm is being injected into an egg during an ICSI procedure
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 (ICSI Video) . 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 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.
ICSI has now become the preferred method of achieving in vitro fertilisation in our clinic. This reduces the risk of unexpected total fertilisation failure sometimes seen with IVF ( research has shown that up to 25% of patients with "unexplained " infertility with an apparently normal semen analysis may have dysfunctional sperm which cannot fertilise eggs in vitro).
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).
For some patients with severe testicular failure, sometimes, it is not possible to find any sperm at all as 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, but the results have been disappointing so far. Other labs are trying to develop methods of in vitro spermatogenesis, in order to mature the spermatids in vitro.
For men with no testis 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 unfertilised egg (the nucleus of which has been removed) and then activating it by electrofusion. While cloning has been performed successfully in many animal species, it has never been used for treating humans so far.
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