**2. Procedural effects of ICSI technique**

The risk of oocyte damage by the ICSI procedure is low in humans and is due to both the skill of the person performing the injection procedure and the quality of the gametes used

during the procedure [8]. The embryologist performing ICSI procedure is a significant predictor of fertilization, and laboratory conditions (i.e. incubators, culture of oocytes individually versus grouped) do not affect the rates [9]. When fertilization failure in most or all of the injected oocytes occurs, with experienced practitioners using normal sperm, the diagnosis falls to oocyte dysfunction, oocyte activation failure, or inability of sperm to be decondensed and processed by the oocyte.

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 119

**Figure 2.** First row: A is an egg with1 pronucleus (PN), B with 3 and C with 4 PN. All these are abnormal fertilizations. Second row: Oocyte in D, E or F, each has 2 PN. This is a sign of normal

Proper orientation of the polar body and needle position are also important, since improper positioning can damage or disrupt the metaphase plate during needle entry. In addition, disturbances in the nuclear spindle may dispose oocytes to aneuploidy or maturation arrest. Thus, perturbation of the cytoskeletal integrity of oocyte may critically influence the fate of the embryo. During ICSI, the location of the first polar body is commonly used as an indication of the spindle position, with the assumption that they are located in close proximity. To avoid damage to the spindle, oocytes are injected at the 3 o'clock position with the first polar body at the 6 or 12 o'clock position. However, with the aid of the computer assisted polarization microscopy, some reports suggest that the location of the first polar body does not necessarily correspond to the spindle position [11, 12]. The reasons for the displacement of the spindle are not fully understood [13]. Further detail on this

Injection of a motile sperm without immobilization leads to poor fertilization rates [14]. In such cases, sperm with moving tails can be seen in the oocyte and sperm-oocyte interaction is obstructed by the normal sperm plasma membrane. Damage to the sperm membrane is necessary for successful oocyte activation following ICSI, as it induces gradual disruption of other parts of the sperm membrane allowing entry of sperm nucleus decondensing factor of the oocyte to induce initial swelling of the head. Because of this swelling, the sperm plasma membrane ruptures and sperm-associated oocyte activating factors are released into the ooplasm and induce oocyte activation. A modified ICSI technique is characterized by

fertilization.

aspect is given under use of Polscope.

Although ICSI is now a routine, it remains a very demanding technique to master, due partly to its inherent technical difficulty and partly to the heterogeneity of the cases. It is generally agreed that the ICSI procedure is subject to a "learning curve" [9] and that one common technical failure is not depositing the sperm within the oocyte cytoplasm. In this situation, the oocyte membrane may not have been broken during attempts to aspirate the ooplasm into the ICSI needle. Thus, the sperm is deposited next to the membrane so that when the oolemma returns to its original position, the sperm is pushed out into the perivitelline space, or is trapped inside a sac formed by the membrane [10]. The sperm may also become adherent to the tip of the injection needle or remain within the injection needle and be inadvertently pulled out upon withdrawal of the needle from the cytoplasm. Aspiration of the ooplasm is always used to make sure that the oocyte membrane is broken during injection. However, if the ooplasm is aspirated too much, degeneration of the oocyte frequently results. The degeneration of oocytes after ICSI is often a result of a fault in the ICSI technique, e.g. an injection pipette that is too large, not positioned properly or not sharp enough. **Figure 1** shows different stages of egg maturation and damaged oocytes after ICSI and **Figure 2** shows normal and abnormal fertilization after ICSI.

**Figure 1.** First row: A and B are GV, C is MI and D is MII oocyte. Second row: E shows typical funnel that appears after ICSI, F shows leakage of ooplasm after ICSI, G shows oocyte damage during denudation and H is an atretic oocyte after ICSI.

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 119

decondensed and processed by the oocyte.

denudation and H is an atretic oocyte after ICSI.

during the procedure [8]. The embryologist performing ICSI procedure is a significant predictor of fertilization, and laboratory conditions (i.e. incubators, culture of oocytes individually versus grouped) do not affect the rates [9]. When fertilization failure in most or all of the injected oocytes occurs, with experienced practitioners using normal sperm, the diagnosis falls to oocyte dysfunction, oocyte activation failure, or inability of sperm to be

Although ICSI is now a routine, it remains a very demanding technique to master, due partly to its inherent technical difficulty and partly to the heterogeneity of the cases. It is generally agreed that the ICSI procedure is subject to a "learning curve" [9] and that one common technical failure is not depositing the sperm within the oocyte cytoplasm. In this situation, the oocyte membrane may not have been broken during attempts to aspirate the ooplasm into the ICSI needle. Thus, the sperm is deposited next to the membrane so that when the oolemma returns to its original position, the sperm is pushed out into the perivitelline space, or is trapped inside a sac formed by the membrane [10]. The sperm may also become adherent to the tip of the injection needle or remain within the injection needle and be inadvertently pulled out upon withdrawal of the needle from the cytoplasm. Aspiration of the ooplasm is always used to make sure that the oocyte membrane is broken during injection. However, if the ooplasm is aspirated too much, degeneration of the oocyte frequently results. The degeneration of oocytes after ICSI is often a result of a fault in the ICSI technique, e.g. an injection pipette that is too large, not positioned properly or not sharp enough. **Figure 1** shows different stages of egg maturation and damaged oocytes after

ICSI and **Figure 2** shows normal and abnormal fertilization after ICSI.

**Figure 1.** First row: A and B are GV, C is MI and D is MII oocyte. Second row: E shows typical funnel that appears after ICSI, F shows leakage of ooplasm after ICSI, G shows oocyte damage during

**Figure 2.** First row: A is an egg with1 pronucleus (PN), B with 3 and C with 4 PN. All these are abnormal fertilizations. Second row: Oocyte in D, E or F, each has 2 PN. This is a sign of normal fertilization.

Proper orientation of the polar body and needle position are also important, since improper positioning can damage or disrupt the metaphase plate during needle entry. In addition, disturbances in the nuclear spindle may dispose oocytes to aneuploidy or maturation arrest. Thus, perturbation of the cytoskeletal integrity of oocyte may critically influence the fate of the embryo. During ICSI, the location of the first polar body is commonly used as an indication of the spindle position, with the assumption that they are located in close proximity. To avoid damage to the spindle, oocytes are injected at the 3 o'clock position with the first polar body at the 6 or 12 o'clock position. However, with the aid of the computer assisted polarization microscopy, some reports suggest that the location of the first polar body does not necessarily correspond to the spindle position [11, 12]. The reasons for the displacement of the spindle are not fully understood [13]. Further detail on this aspect is given under use of Polscope.

Injection of a motile sperm without immobilization leads to poor fertilization rates [14]. In such cases, sperm with moving tails can be seen in the oocyte and sperm-oocyte interaction is obstructed by the normal sperm plasma membrane. Damage to the sperm membrane is necessary for successful oocyte activation following ICSI, as it induces gradual disruption of other parts of the sperm membrane allowing entry of sperm nucleus decondensing factor of the oocyte to induce initial swelling of the head. Because of this swelling, the sperm plasma membrane ruptures and sperm-associated oocyte activating factors are released into the ooplasm and induce oocyte activation. A modified ICSI technique is characterized by pushing the needle tip close to the membrane opposite the puncture site, aspirating the cytoplasm at this point and releasing the sperm in the centre of oocyte [5]. This modification improves fertilization in oocyte-dependent activation failure, but its routine application does not improve the overall results.

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 121

with high power Nomarski optics enhanced with digital imaging. Such examination helps to identify spermatozoa with a normal nucleus and nuclear content [19]. The exact indications of IMSI [20] and usefulness [21] are debatable. Finding normal-looking spermatozoa took a minimum of 60 min, and up to 210 min, depending on the quality of the semen sample. The technique required two embryologists working together on the analysis of the same sample at the same time in order to minimize the subjective nature of sperm evaluation [22]. The IMSI procedure improved embryo development and the laboratory and clinical outcomes of sperm microinjection in the same infertile couples with male infertility and poor embryo development over the previous ICSI attempts [23]. A successful childbirth after IMSI without assisted oocyte activation in a patient with globozoospermia has been reported [24]. Randomized large-scale trials are needed to confirm the beneficial effects of IMSI in couples

The oocyte spindle can be imaged non-invasively based on the birefringence, an inherent optical property of highly ordered molecules, such as microtubules, as they are illuminated with polarized light. Polarized light microscopy has been applied to embryology for decades. A digital, orientation-independent polarized light microscope, the Polscope, has demonstrated the exquisite sensitivity needed to image the low levels of birefringence exhibited by mammalian spindles [26]. The Polscope, is used to protect the meiotic spindle from damage during ICSI. The oocytes having Polscope visualised spindle have higher fertilization rate. When the spindle is located at 0°-30° in relation to the first polar body, ICSI achieves highest fertilization rate [27]. The use of Polscope is still not widely practiced and further improvements are needed. Morphometric evaluation of the spindle through the Polscope is not consistent with confocal analysis. This suggests that the Polscope may still be

Both nuclear and cytoplasmic maturation of oocytes have to be completed to ensure optimal conditions for fertilization. Oocytes are retrieved prior to ovulation for IVF or ICSI procedures. In the pre-ovulatory phase, meiotic division of the oocyte must progress to metaphase II which is considered nuclear maturation and is evident by extrusion of first polar body. The oocyte also must develop the capacity to attain fertilization and initiate embryo development which is considered cytoplasmic maturation [29, 30]. Cytoplasmic maturity is thought to be asynchronous with nuclear maturity in stimulated cycles [31, 32] and the fertilizing ability of an oocyte with a mature nucleus is not necessarily at its maximum potential. Thus, preincubation of oocytes prior to IVF or ICSI may induce cytoplasmic maturation that could eventually increase fertilization and also pregnancy rates. The human oocytes progressively develop the ability for full activation and normal

a rather inefficient method for assessing the metaphase II spindle [28].

with poor reproductive prognoses [25].

**5. Use of polscope** 

**6. Timing of ICSI** 

development during the MII arrest stage [33].
