Trophectoderm vesicles (TVs) are observed in some blastocysts that penetrate cells from the zona pellucida to the outer margin. Therefore, we compared this incidence in relation to hatching, pregnancy, and miscarriage rates between conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI). Vitrified/warmed blastocysts (n = 112) were derived from surplus embryos. The blastocysts were then observed using time-lapse cinematography to resolve the relationship between hatching and implantation. Another study was conducted that comprised 681 embryo transfer cycles in 533 patients who received a single vitrified/warmed blastocyst from our clinic. The incidence of TV was significantly higher in embryos inseminated by ICSI compared with c-IVF [ICSI: 51/56 (91 %)
c-IVF: 25/56 (45 %)
P &lt
 0.01]. The successful hatching rate was significantly lower in ICSI than in c-IVF [ICSI: 11/56 (20 %)
c-IVF: 29/56 (52 %)
P &lt
 0.01]. In addition, the hatching rate was significantly lower when TVs were present (14/76
18 %) than in non-TV embryos (26/36
72 %) (P &lt
 0.01). In regard to the clinical study results, no significant differences were found between the groups in the pregnancy rate (TV present group: 107/183, 58.5 %
TV absent group: 273/498, 54.8 %) and miscarriage rate (TV present group: 21/107, 19.6 %
TV absent group: 53/273, 19.4 %). In vivo, we hypothesized that hatching and hatched would occur naturally by assisting protease action in the uterus
therefore, these results suggest that the presence of TV has no effect on pregnancy rates in the clinical setting.