Intracytoplasmic sperm injection (ICSI) has wide clinical application. In order to achieve good results with this method, it is important to restrict the possibility of oocyte injury as much as possible, and securely inject spermatozoa into the ooplasm. For this purpose, we clinically applied piezo-ICSI, which employs a micromanipulator with piezoelectric elements, to humans, and compared the results with those obtained by conventional ICSI. Conventional ICSI and piezo-ICSI were used in 279 cycles and 335 cycles respectively. Piezo-ICSI showed significantly more favourable results, with a survival rate of 88.1% (conventional ICSI: 81.4, P < 0.001), a fertilization rate of 79.4% (conventional ICSI: 66.4%, P < 0.001), and a pregnancy rate of 23.1% (conventional ICSI: 14.9%, P < 0.05). In piezo-ICSI, the needle used is not sharpened and has a flat tip. However, deformation of the oocyte during insertion of the needle is restrained by vibration of the piezo, and the oolemma is punctured readily and securely by the piezo pulse, at the site where the spermatozoon is injected. Piezo-ICSI is a promising new technique for human ICSI that should improve the survival, fertilization and pregnancy rates after ICSI.

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In a total of 1048 intracytoplasmic sperm injection (ICSI) cycles, motile spermatozoa from four out of 424 patients (0.9%) failed to fertilize oocytes, despite an apparently successful ICSI procedure. No activation was observed in these injected oocytes. The spermatozoa from three of the four patients were injected into unfertilized mouse oocytes by ICSI (mouse test) to evaluate their oocyte activating ability. The oocyte activation rate of the spermatozoa of patients A, B, and C in the mouse test was 46, 100, and 86% respectively (control: 100%). Simultaneous injection of two spermatozoa from patient A into the mouse oocytes increased the oocyte activating rate to 89% (sham control: 29%). 100% fertilization rates were obtained for patients A and B by combining ICSI and electrical stimulation, and this resulted in pregnancy and the birth of healthy twins for the partner of patient A. Thus, it is considered that the spermatozoa of these patients are not lacking sperm factors but that the activity of these factors is depressed. The combination of ICSI and electrical stimulation is effective in these cases.

Oocyte activation and Ca2+ oscillation-inducing abilities of round spermatid (ROS) and elongated spermatid (ELS) of some rodents and human were assessed by their injection into mouse (B6D2F1) oocytes (mouse test). With mice (B6D2F1, ICR) and rat, ROS displayed no oocyte activation or Ca2+ oscillation-inducing abilities. Although ELS could induce activation at 87, 86 and 31% of injected oocytes respectively, most of the intracellular calcium concentration ([Ca2+]i) responses of ELS-injected oocytes did not show oscillation patterns; only several transient [Ca2+]i rises (transient pattern) were seen. Similarly, with hamster, rabbit and human, while ROS could induce oocyte activation efficiently (70, 71 and 52% respectively), most of the [Ca2+]i patterns of injected oocytes were transient patterns, and not oscillation patterns. When ROS nuclei only from these latter species were injected into mouse oocytes, most of the oocytes could not be activated. [Ca2+]i patterns of oocytes injected with immature sperm cells changed from transient pattern to oscillation pattern while the cells were maturing into spermatozoa. With hamster ROS, oocyte-activating factor was found to be distributed mainly in the cytoplasm. It was interesting that there is a dissociation between the timings of appearance of oocyte activation and that of Ca2+ oscillation of oocytes injected with developing immature sperm cells.

Fertilization failure (complete fertilization failure or low fertilization rates) after intracytoplasmic sperm injection (ICSI) can occur in rare cases. In the majority of these cases, the unfertilized oocytes are inactivated. Assisted oocyte activation was applied as a treatment option for a case of low fertilization rate as a clinical trial. A patient with a low fertilization rate (ranging from 0% to 33.3%; mean = 17.0%) after eight previous ICSI cycles at another hospital, was diagnosed with fertilization failure. The most likely cause of fertilization failure was failure of oocyte activation. Therefore, artificial oocyte activation by strontium treatment was combined with ICSI to achieve viable fertilized oocytes. Oocytes were stimulated with strontium (10 mM SrCl(2), 60 min) approximately 30 min after ICSl. Six injected oocytes were stimulated and all were then successfully fertilized. Two blastocysts were transferred into the uterus, resulting in a pregnancy and birth. A second pregnancy was achieved following implantation of two cryopreserved embryos (one blastocyst and one morula). In conclusion, strontium treatment was found to be an effective method for artificial oocyte activation in a case with a low fertilization rate after ICSI.