**3. IMSI in practice**

The freshly ejaculated semen is centrifuged at approximately 360 ×*g* for 10 min. In cases when sperm concentration is <5 × 106 , centrifugation is performed for 1520 min. The pellet is resuspended in 0.5 ml of sperm wash solution (Sperm Preparation Medium, Origio, Denmark). Further assessment is performed by using the bilayer density gradient: 0.5 ml of 50% gradient solution and 0.5 ml of 80% (Supra Sperm Origio, Denmark) for 15 min. In severe oligospermic cases, 0.3 ml of each of the gradient layers is used and centrifugation in 300 × *g* should last up to 1 h [39, 40]. Final sperm pellet is resuspended in 50–100 μl. Approximately a droplet of 1 μl is placed inside the observation droplet of the pre-prepared IMSI dish. During the observation, motile spermatozoa with rough morphological abnormalities are omitted and not aspirated. Other motile sperm cells are aspirated and evaluated inside the ICSI pipette or in the clean PVP droplet and scored as mentioned above. The individual scored sperm cell is then placed into the appropriate droplet, in the IMSI dish [32, 35]. Aspiration of sperm cells is performed by the means of micromanipulations. There is no preference for a certain micromanipulation system. One should work with a system which is the easiest to handle, with his own hands.

#### **3.1. Choosing the sperm**

*Clean droplet of PVP 10%*: droplets of 1 μl are located in parallel to the droplets with the spermatozoa. When the intensity of the processed spermatozoa is not high, the authors like to create tiny "bridges" between these two droplets. The tiny bride will allow the passage of spermatozoa to the 10% PVP droplet and it eases the detection on the spermatozoa, as motility of the examined spermatozoa is slowed down. It is recommended to draw tiny extensions from the rim of the PVP droplets to capture the heads of the motile sperm cells. All droplets should be covered by mineral culture oil, which is generally used for the ICSI procedure.

The authors recommend that based on their existing design of the IMSI dish, each laboratory should adapt its special design for the arrangement of the droplets, in accordance with the

The freshly ejaculated semen is centrifuged at approximately 360 ×*g* for 10 min. In cases when

pended in 0.5 ml of sperm wash solution (Sperm Preparation Medium, Origio, Denmark). Further assessment is performed by using the bilayer density gradient: 0.5 ml of 50% gradient

, centrifugation is performed for 1520 min. The pellet is resus-

preference of the laboratory where the ICSI procedure takes place.

**3. IMSI in practice**

**Figure 1.** Diagram of an IMSI dish.

206 Spermatozoa - Facts and Perspectives

sperm concentration is <5 × 106

The group of Bartoov classified the selected spermatozoa morphology as "normal" and "second" choice, based on the data collected by scanning and transmission electron microscopy. Some characteristics of the morphology of the head of the sperm cell such as smoothness, symmetry, oval configuration, average length and width (4.75 ± 0.28 and 3.28 ± 0.20 mm, respectively), and nuclear chromatin mass containing no more than one vacuole were defined for normalcy of the sperm nucleus [32, 33]. The "second-choice" motile sperm was not clearly depicted. One might only speculate that during IMSI, when no normal head spermatozoa can be found, the alternative then is to select motile sperm cells that are morphologically the second-best choice.

The Cassuto and Barak Score was developed in order to define more precisely the preferable spermatozoon that should be injected into the oocyte, in real time [35]. This scoring system was established after checking and taking into consideration the sperm defects, which negatively affect the development of the embryo in vitro and had the name of HAVBIC: **H**ead: normalcy and shape, **A**crosome: presence or absence, **V**acuoles: presence or absence, **B**asis of the sperm head, **I**nsertion: the axial position of the sperm, and **C**ytoplasmic droplet: presence or absence. Logistic regression was used for fertilization and embryo development as dependent variables. Coefficients were calculated and tested by comparing receiver operating characteristic (ROC) curves. The best results were achieved with an area under the curve of 0.618, deriving the following formula: Score of spermatozoa = 2 × Head + 3 × Vacuole + 1 × Base. Following these findings, this classification system took into consideration three major parameters of the sperm nucleus: normalcy of shape and size of sperm head, lack of vacuoles and normalcy of the base of the head of the specific spermatozoon. Each of the normal parameter received a value = 1 when normal or = 0 when abnormal. Classification of the individual sperm cell, with a maximum of score 6, is calculated by the embryologist, who performs the IMSI, with this friendly formula, described above, and distinguishes between three classes:

Class 1: spermatozoa of highest quality, score 4–6; class 2: spermatozoa, score 1–3; class 3: low-quality spermatozoa, score 0. In their study a difference was noted between the fertilization rate of oocytes with regard to the classification of the injected spermatozoa (*P* < 0.04; chi square = 6.31). A pair-wise comparison showed a higher fertilization rate in oocytes injected with class 1 spermatozoa in comparison to class 3 (*P* < 0.01; chi square = 6.3). A difference was noted in the development rate into expanded blastocyst (*P* < 0.03; chi square = 6.71), no expanded blastocyst was observed in embryos resulted from injection of class 3 spermatozoa (score of 0) [35].
