Occasionally, spermatozoa fail to fertilize even when they are artificially placed in close proximity to eggs during conventional in vitro fertilization (IVF). This is particularly common if semen parameters are abnormal, or when the number of spermatozoa is insufficient. Intracytoplasmic sperm injection (ICSI) is a technique used to inject individual sperm into an egg. The procedure was developed in 1992 in Belgium by Palermo and colleagues and due to its dramatic progress has revolutionized the treatment of male factor infertility.
Until 1992, most infertility failures originating from severe male factor were untreatable. Micromanipulation techniques, such as partial zona dissection (PZD) and subzonal sperm injection (SUZI), designed to overcome the poor performance of sperm cells, did not substantially improve the success rate of in vivo fertilization.

Laboratory Procedures

Technical procedure
The ICSI procedure involves the introcytoplasmatic injection of a single sperm cell into an egg. Furthermore, as the sperm is microinjected into the ooplasm, it bypasses the passage through the zona pellucida and is not required to interact with the oolemma. Therefore, infertility problems that originate from faulty sperm-egg interaction may also be resolved by this IVF treatment protocol.


Handling of oocytes
According to all treatment protocols, identification of the cumulus-oocyte complexes and evaluation of their maturity (i.e., score for mature, slightly mature, completely immature, or slightly hypermature) are performed immediately after follicle aspiration. Unlike conventional IVF, in which intact mature cumulus-oocyte complexes are inseminated, cumulus cells that surround the eggs are removed before microinjection. Denudation of the mature oocytes (removal of the cumulus corona cells) is an essential prerequisite for ICSI. Removal of the surrounding cumulus cells is accomplished by a combined enzymatic (exposure to HTF-HEPES-buffered medium containing 80 IU/ml of hyaluronidase) and mechanical treatment (the oocytes are repeatedly aspirated through a hand-drawn Pasteur pipette with an inner diameter of ~200mm) carried out under a stereoscopic dissecting microscope.
The removal of the serounding cells  is necessary for observation of the oocyte, Cumulus cells may block the injecting needle, thus interfering with oocyte microinjection.
Each oocyte is then examined under the microscope to assess the maturation stage and its integrity; metaphase II (MII) is assessed according to the absence of the germinal vesicle (GV) and the presence of an extruded polar body. ICSI is performed only in oocytes that have reached MII of maturity (absence of the GV and presence of the first polar body). 

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Oocyte with cumulus
after aspiration Oocyte prepared for ICSI after removal of the cumulus cells

Working table of the microscope

Handling of the Spermatozoa
The spermatozoa are observed while they are in motion, and are selected under magnification x400, according to morphologic characteristics, such as shape, light refraction, and motion pattern in the viscous medium. Preference is given to spermatozoa that swim at the droplet edge.
Once selected, the sperm is immobilized by breaking its tail, which is achieved through gentle mechanical pressure. The procedure is performed when the spermatozoa are positioned at a 90° angle to the tip of the pipette, which is then lowered gently to compress the sperm flagellum. This is accomplished by dragging the injection needle across the tail until a visible kink or break can be seen. A correctly immobilized sperm should maintain the shape of its tail. The immobilized sperm is then aspirated into the needle. Immature gametes probably require additional manipulation to promote membrane permeabilization that enhances the postinjection events involved in nuclear decondensation (these changes are related to important modifications in the nuclear chromatin and several tail organelles during the epididymal transit). These modifications include the formation of disulfide bonds, a change in the membrane surface charge, a profound qualitative and quantitative modification in their lipid composition, and the absorption of specific proteins secreted by the epithelium of the epididymis). Lack of all these changes is associated with a decreased ability of an epididymal sperm to bind and penetrate the oocyte.

Injecting the sperm into the egg
The oocyte is held in place by suction applied to the holding pipette while the injection pipette is lowered and focused in alignment with the outer right border of the oolemma on the equatorial plane at the 3 o’clock position. The injecting pipette is pushed against the zona, permitting its penetration and thrusting forward to the inner surface of the oolemma. As the point of the pipette reaches the approximate center of the egg, a break occurs in the membrane. This is reflected by a proximal flow of the cytoplasmic organelles and the spermatozoon back up into the pipette. The sperm is then slowly ejected back into the cytoplasm, where aspiration of the cytoplasm becomes an additional stimulus to activate the egg. Once the pipette is removed, the breach area is observed, and the order of the opening should maintain a funnel shape with the vertex into the egg.

Aspirating the sperm into the injecting pipette

The micromanipulator is a device used to interact with the oocyte and the sperm under a microscope, where a level of precise movement is necessary so that it cannot be achieved by the unaided human hand. This apparatus consists of an input joystick, a mechanism for reducing the range of movement, and an output section by means of grasping a microtool to hold the oocyte and inject the sperm as required.
This process is essential with the ICSI procedure as the size of the spermatozoon is approximately 3 to 5 microns and it is injected into an oocyte approximately 100 microns in diameter. This procedure is performed under the direct manual control of an embryologist. 
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The micromanipulator and working
Procedure for injecting the sperm into the oocyte
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Injecting the sperm into the oocyte

Summary of the ICSI process


Sperm-related issues:
Oligoteratoasthenozoospermia (OTA) syndrome, i.e., low sperm count, decreases sperm motility, and abnormal sperm morphology (size and shape).

Other sperm abnormalities
Recurrent pyospermia
Collection of sperm from the testes
Frozen-thawed sperm

Oocyte-related issues
Small number of retrieved eggs
Use of cryothawed oocytes
Oocytes considered for preimplantation genetic diagnosis (PGD). This procedure is usually performed to avoid DNA contamination from the many spermatozoa attached to the cumulus cells and zona pellucida.

Embryo-related issues
When an embryo is needed for gene defect analysis

Female-related issues
Advanced age

Male-related issues:
Ejaculatory dysfunction
Retrograde ejaculation

General issues
Failed fertilization by regular IVF
Unexplained infertility
Immunological infertility

Concerns Related to the ICSI Procedure

The procedure

During piercing of the cell membrane caution should be taken not to damage the inner DNA structure.
This is accomplished by orienting the oocyte so that the polar body is placed in a 900 position to the entrance of the injecting pipette.
The injection procedure can introduce foreign material into the oocyte, such as culture medium, seminal fluid with or without bacteria, viruses, or in theory, even prions or foreign DNA, in addition to male mitochondria, acrosomal cap, perinuclear theca, all of which are normally lost as the sperm penetrates the shell of the oocyte. Therefore, the ICSI process of fertilization is somewhat different from the usual IVF.








Results of the ICSI procedure in terms of healthy pregnancy
During the ICSI procedure, a small number of eggs (usually <5%) can be irreparably damaged as a result of the ICSI needle insertion.
There is no evidence that the miscarriage rate of ICSI and IVF pregnancies is similar.
The incidence of prematurity and low birth weight babies is similar to that for IVF, but slightly higher than rates found in natural pregnancies.
The overall risk of having a baby with a chromosomal abnormality in the X or Y chromosomes is 0.8%, or 8 per 1000 (this risk is four-fold higher than the average associated with spontaneous conception). This can be related to sex chromosome abnormalities: increased risk of miscarriage; affected infants could be afflicted with heart problems that may require surgery; increased risk of behavior or learning disabilities in affected children, and higher risk of infertility of these children in adulthood.

It appears that children conceived either by ICSI or IVF have an increased odds ratio (2.77 and 1.8, respectively) for malformations that need medical or surgical intervention in early life compared to naturally-conceived children.
According to the Bayley and other intelligence scales, ICSI children performed normally in psychological testing as well as in their cognitive and verbal skills.

Follow-up of children up to 10 years of age
In a study published in the Human Reproduction Journal, Leunens and colleagues followed-up ICSI-conceived children up to 10 years of age. Leunens et al. concluded that these children achieved total, verbal and performance intelligence scores comparable to those of spontaneously-conceived children. No significant differences were found in a comparison of overall motor, manual and ball skills. Regarding long-term follow-up, the IQ results of ICSI children and the spontaneously-conceived children at age 10 appear to have converged (from slightly higher scores in the ICSI children at age 8), probably indicating a decreased effect of maternal educational level, or stimulating home environment in the ICSI group over time.

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