A NOVEL ZONA PELLUCIDA (ZP)-FREE CULTURE SYSTEM FOR SEVERE CYTOPLASMIC FRAGMENTATION CASES: A PILOT STUDY USING 3PN EMBRYOS AND TIME-LAPSE CINEMATOGRAPHY

Abstract

A study in 2017 observed perivitelline threads in more than 50% of cleavage-stage human embryos using time-lapse imaging, and the rate of cytoplasmic fragmentation (at the first cleavage) was significantly decreased in embryos without perivitelline threads (P < 0.001). While it is proposed that perivitelline threads play an important role in crosslinking the cumulus cells and oocyte during maturation, the mechanism underlying such a role remains unclear. It is also unknown whether the threads still function in mature MII oocytes. Therefore, in this study, zona pellucida of abnormally-fertilized oocytes which were donated by patients was removed at pronuclear stage. Those ZP-free oocytes were observed in time-lapse culturing system in order to examine developmental morphology. This study used 57 abnormally fertilized (3PN) embryos (cIVF: n = 51, ICSI: n = 6) donated by assisted reproduction technology patients in our clinic with informed consent since 2017. After confirming the three pronuclei, we removed the ZP from each 3PN embryo using a laser, and the resultant ZP-free embryos were cultured and observed in an incubator equipped with a time-lapse imaging system. For ZP removal, 3PN embryos were placed in drops of 0.125M sucrose-containing HEPES media that had been covered with mineral oil and warmed to 37°C. Despite a small reduction in ooplasm size, half of the ZP was removed by laser (Saturn 5; Origio、Lykos; Hamilton Thorne). Subsequently, the ooplasms were completely separated from their ZPs by pipetting, and these ZP-free 3PN embryos were cultured continuously for 5 days with time-lapse imaging. Of 58 ZP-free embryos in total, 54 (94.7%) were cleaved, and there was no significant decrease in cleavage rate compared to 2PN embryos (98%) used routinely in our clinic. Furthermore, 28 of the 54 embryos (51.9%) developed to the morula stage after third cleavage, and 18 embryos (33.3%) formed a blastocoel and became blastocysts. Thus, removing the ZP before cleavage did not adversely affect the embryo development. In terms of the amount of fragmentation, based on the modified Veeck’s criteria, 36 of 54 ZP-free 3PN embryos (66.7%) showed less than 20% of the volume in fragments compared to the total volume of cytoplasm at the first cleavage (Grade 1 and 2), 14 (25.9%) showed 20-40% fragments (Grade 3), and only 4 (7.4%) showed > 40% fragments (Grade 4). These results suggested that the rate of fragmentation was decreased by ZP removal before the first cleavage. This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the ZP-Free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation.