Study question Is the implantation rate (IR) higher in blastocysts with trophectodermal vesicles (TVs) compared to blastocysts without TVs or euploid blastocysts with unknown spontaneous hatching status? Summary answer The blastocysts with TVs demonstrate significantly higher IR in comparison to blastocysts without TVs or euploid blastocysts with unknown spontaneous hatching status. What is known already After ICSI spontaneous hatching mainly occurs by trophectoderm cell herniation via a small slit in the zona pellucida. At the beginning of this process, TVs are formed on the outside of the zona pellucida. It was previously shown that the clinical pregnancy rate was similar after the transfer of expanded blastocysts and expanded blastocysts with TVs. But another study showed that transfer of blastocysts of more advanced hatching stages yields better pregnancy rates than expanded blastocyst transfer. It remains unclear whether there is an association between the presence of TVs and IR in the transfers of single vitrified blastocysts. Study design, size, duration This retrospective cohort study was conducted from October 2018 to November 2019 and included 477 transfers of a single vitrified blastocyst. Cases were divided into 3 groups. Group 1 included transfers of blastocysts without TVs and with assisted hatching (AH). Group 2 contained the transfers of blastocysts at TVs stage of spontaneous hatching and without AH. Group 3 consisted of transfers of the euploid blastocysts with AH performed and unknown spontaneous hatching status. Participants/materials, setting, methods The age of women was between 21 and 39. Embryo transfers following oocyte donation programs were excluded from the study. This study included only transfers of the ICSI-derived fully expanded blastocysts with top-graded inner cell mass and trophectoderm. AH was performed using laser Saturn 3. The primary outcome was the implantation rate. Statistical analysis was performed using Pearson’s chi-square test and likelihood ratio test. Preimplantation genetic testing for aneuploidy (PGT-A) was performed by next-generation sequencing. Main results and the role of chance The number of cases in groups 1,2 and 3 was 133, 49, and 295, respectively. The average age in the groups was about 32.5 and did not differ between groups. The implantation rate in group 3 with PGT-A was 60% (177 out of 295), which was insignificantly higher compared to group 1 - 55% (73 out of 133) (p = 0.34). In group 2, the implantation rate was 76% (38 out of 49), which exceeded significantly the outcomes in groups 1 and 2 (p = 0.016). Thus the transfer of expanded blastocyst with TVs gives higher IR in comparison to expanded blastocyst. Therefore TVs could be utilized as a morphological marker for embryo selection. Furthermore, according to obtained results the presence of TVs on nontested blastocysts predicts implantation better than euploidy does in blastocysts with unknown spontaneous hatching status. Limitations, reasons for caution This is a retrospective nonrandomized study with its inherited limitations. Wider implications of the findings: Based on the results of the study embryo selection practice could be optimized. To maximize the outcomes of PGT-A programs embryo culture and biopsy workflow could be modified to allow collecting data on spontaneous hatching and TVs presence before performing the biopsy. Trial registration number Not applicable