**5. IMSI and head-sperm vacuoles**

Vanderzwalmen et al., classified the spermatozoa according to the presence and size of vacuoles into four groups: Grade I: normal shape and no vacuoles; Grade II: normal shape and maximum of two small vacuoles; Grade III: normal shape and more than two small vacuoles or one large vacuole; and Grade IV: large vacuoles in conjunction with abnormal head shapes or other abnormalities at the level of the base of the sperm head. The outcome of embryo development in a group of 25 patients after sibling oocyte injection with the four different grades of spermatozoa showed no significant difference in embryo quality up to day 3. However, the occurrence of blastocyst formation was 56.3 and 61.4% with grade I and II spermatozoa, respectively, compared with 5.1% with grade III and 0% with grade IV, respectively (*P* < 0.001) [44]. It is not clear yet why presence of vacuoles in the sperm head is such an important parameter of sperm quality. Some reports showed no correlation between the appearances of vacuoles to male infertility [45–47]. One of these studies was an unpowered investigation [47] and another evaluated the sperm under magnification of 1000× [45]. Many others reported that vacuoles might negatively be associated with male fertility potential [32–37, 44, 48–54]. Moreover, investigation of the relation between sperm vacuoles and acrosome reaction suggested that there might be a negative link between presence of vacuoles and acrosome reaction of the sperm [55, 56]. Consequently, IMSI could be a method for assisting the removal of the acrosome reaction-resistant spermatozoa.

The personal attitude of the authors of the current dissertation is that majority of abnormalities observed under the high magnification of IMSI in real time are probably not visible while using routine conditions with lower magnifications. It is likely, though, that the benefit of scoring scale of the sperm cell is a kind of "fine tuning"; IMSI therefore is more beneficial for motile spermatozoa which have normal morphological appearance under magnification 200–400×.
