In 2011, Istanbul consensus workshop has proposed that the assessment of syngamy (24 h post insemination) is very sensitive predictors for embryonic quality (RBM Online, 2011). However, the value as independent predictor of outcome is still unclear. We have reported several novel aspects of human embryonic development in vitro using time-lapse cinematography (TLC). In this study, we analyzed the relationship between timing of syngamy and human embryonic development using TLC. We have developed a system of TLC, which is described elsewhere (AJOG, 2008). Culture temperature was maintained at 37.0 ± 0.2°C and pH at 7.37 ± 0.05 by controlling CO2 flow. After c-IVF or ICSI procedure, oocytes donated (n=203) were analyzed for TLC. In c-IVF, the cumulus cells were gently removed at one hour post insemination, so as not to damage the tail of the sperm that had penetrated the zona pellucida, and transferred into culture media for TLC. In ICSI, oocytes were commenced TLC after the procedure. Of 203 oocytes, normally fertilized embryos were 128 (c-IVF: 50, ICSI: 78) and the rest were abnormal fertilization (n=13), undetectable (n=35) and unfertilized (n=27). In normal fertilized embryos, the time required from the extrusion of 2nd polar body to syngamy in c-IVF and ICSI were 21.2 ± 3.6 hours and 20.8 ± 3.6 hours, respectively. The time required for syngamy in good quality embryos (GQE) was 20.0 ± 3.3 hours and in poor quality embryos (PQE) was 22.1 ± 4.3 hours, respectively and the difference was significant (P<0.05). In addition, the time required for syngamy in abnormally fertilized embryos was 25.1± 6.3 which was obviously delayed compare to normal fertilized embryos. Although there was no difference in the time required for syngamy by the insemination procedures (IVF or ICSI), the time required for syngamy in GQE was significantly shorter than that in PQE, suggesting that timing of syngamy would be a useful parameter to assess the embryo quality in human embryos. Furthermore, the time required for syngamy between normally and abnormally fertilized embryos was significantly different, suggesting that we could distinguish the normally fertilized embryos from abnormal embryos by assessment of timing of syngamy.

To analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time-lapse cinematography. We had 122 oocytes donated for this study from couples that provided informed consent. We recorded human oocytes at 2,000 to 2,800 frames every 10 s during the fertilization process and thereafter every 2 min using a new in vitro culture system originally developed by the authors for time-lapse cinematography. We displayed 30 frames per second for analysis of the polyspermy block during fertilization. Three oocytes showed the leading and following sperm within the zona pellucida in the same microscopic field. The dynamic images obtained during the fertilization process using this new system revealed that once a leading sperm penetrated the zona pellucida and attached to the oocyte membrane, a following sperm was arrested from further penetration into the zona pellucida within 10 s. The present results strongly suggest the existence of a novel mechanism of polyspermy block that takes place at the zona pellucida immediately after fertilization. These findings are clearly different from previous mechanisms describing polyspermy block as the oocyte membrane block to sperm penetration and the zona reaction. The finding presented herein thus represents a novel discovery about the highly complicated polyspermy block mechanism occurring in human oocytes.