**2. Intracytoplasmic morphologically selected sperm injection step by step**

#### **2.1. The principle of the IMSI microscope**

in 1978. This revolutionary assisted reproductive technology (ART) attempt brought the fulfillment of a vision to hundreds of thousands of infertile couples to conceive and deliver. The main idea behind the development of the technique was treating women with mechanical factor infertility. Despite the great success of IVF, poor results were obtained in cases of severe male infertility. IVF outcome was satisfactory when oocytes were inseminated with >1.5 million spermatozoa and at least 50% motility could be observed in the specimen. This major limitation of sperm insemination in routine IVF was overcome by intracytoplasmic sperm injection (ICSI), introduced by Palermo in 1992 [1]. The injection of a single sperm into an oocyte, which revealed fertilization and development of viable in vitro embryos, gave a rapid solution to men with severe forms of male factor infertility, allowed application to other forms of infertility. Nowadays, it serves as the main tool for assisting couples to fulfill their wish to conceive [2]. Furthermore, it is also used for various infertility indications as low number and poor morphology of oocytes, thick zona pellucida, pre-implantation genetic diagnosis or screening (PGD, PGS), cases of hepatitis C and human immunodeficiency virus, in vitro maturation, and oocyte cryopreservation. The usage of ICSI enabled to achieve fertilization, embryo development, pregnancies, and deliveries. It has been reported that strict morphologic criteria do not affect ICSI outcomes [3–9]. On the other hand, scientists indicated that the paternal gamete influences the resulting embryo, mainly at the level of blastocyst formation. Several points of negative paternal impact, both genetic and epigenetic, have been identified in embryos following the injection of poor-quality spermatozoa into the oocyte. Using ICSI also clarified some points of the paternal influence on the embryo development, mainly in vitro [10–20]. The specific injected sperm might affect the health of the newborns [21]. Several years ago, a series of reports pointed out that children born as a result of ART were found to have increased frequencies of a number of diseases known to have an epigenetic etiology [22–26], mainly after ICSI [27, 28]. Recently, Santos et al. evaluated the genome-wide DNA methylation together with chromatin organization in human embryos derived by either IVF or ICSI. In this report it was found that infertility per se, rather than ART procedures, may play an important part in predisposition to epimutations that leads to diseases of an epigenetic basis. Interestingly, embryos developing to the blastocyst stage had an apparently normal epigenotype irrespective of the procedure used. These investigators concluded that ICSI per se does not lead to an increased incidence of epigenetic errors [29]. It is accepted that routine morphology characteristics may not necessarily describe the quality of the specific single spermatozoon that was injected into the oocyte. Semen analyses, which describe the general picture of sperm quality of the raw ejaculate, are based on the examination of fixed and stained sperm cells. The latest, obviously, is not suitable for treatment any more. Aspiration of a specific sperm cell, and its injection into the oocyte, during ICSI, regularly takes place under the magnification of 200× or 400×. The sperm cell is randomly chosen when spermatozoa with major morphological abnormalities are generally omitted due to the experienced eyes of the embryologist. It is questionable as to what tool should be used to evaluate morphological criteria in real time during ICSI, which will enable to select a specific spermatozoon, with the highest predictive value for better fertilization, embryo developmental, implantation and pregnancy. De Vos et al. showed a correlation between the gross morphology of the individual injected sperm cell and the formation and development of the resultant embryo. These scientists admitted

204 Spermatozoa - Facts and Perspectives

The term IMSI was defined by Bartoov et al. [32, 33]. The idea was based on the magnification up to 6000× and more of the motile sperm cell in real time [33, 35–38]. The sperm analysis is performed using an interference phase contrast inverted microscope with the optics of Nomarski. The magnified image of the sperm cell is displayed on a screen. The final magnification of the "on-screen image" is actually a combination of the magnification of the objective, the camera adapter, ratio between the diagonal screen size in mm, diagonal of the camera chip size in mm, and internal magnification of the microscope. Depending upon these specific characteristics of an IMSI system, the final value of the magnification might vary from 6000× to 6600× as described earlier. It should be clarified that the final various magnifications achieved in IMSI are actually a result of zoom on the image of the sperm cell, which magnifies the structures of its surface and cannot be observed in magnification of 200–400×, due to objective limitations of the human eyes.

#### **2.2. Preparation of the IMSI dish**

The high magnification of the objective (100×) with the optics of Nomarski requires a dish with a glass bottom. Droplets of the medium, approximately droplets from 1 to 4 μl, should be located on the dish as described in the diagram of an IMSI dish (**Figure 1**).

In **Figure 1**, the diagram of the IMSI dish describes the arrangement of the observation droplets with spermatozoa, droplets with PVP, droplets of clean medium for the selected spermatozoa, and clean droplets for the injection of the oocytes. The latest should be replaced with fresh droplets of media, prior to the ICSI procedure.

*Observation droplets*: three droplets of 2 μl are located in the left side of the dish. In these droplets, 1 μl of the processed sperm cells of the sperm ejaculate will be inserted. These droplets of the sperm culture medium might contain, in correlation with the intensity of the sperm motility, between 0 and 10% polyvinylpyrrolidone (PVP) solution [34].

*Clean droplets of a clean sperm culture medium*: this is required to host the scored sperm cells after the evaluation. In each droplet, aspirated spermatozoa with a distinct morphological score are located.

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.

Interacytoplasmic Morphologically Selected Sperm Injection: A Tool for Selecting the Best…

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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 alterna-

tive 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].

**3.1. Choosing the sperm**

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

*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 preference of the laboratory where the ICSI procedure takes place.
