Abstract Study question Is it possible to determine the difference between 2.1 pronuclear (2.1PN) zygotes and tripronuclear (3PN) zygotes from time-lapse images? Summary answer A pronucleus of less than 15 μm in diameter can be considered the micro pronucleus (micro PN), and it is possible to classify 2.1PN zygotes. What is known already 2.1PN zygotes are defined as zygotes with two pronuclei and one smaller pronucleus. Capalbo et al. (2017) reported that most of the 2.1PN-derived blastocysts were diploid by preimplantation genetic testing for aneuploidy (PGT-A), including single-nucleotide polymorphisms (SNPs) analysis. Thus, the treatment with 2.1 PN zygotes should be performed with chromosome testing. In Japan, where PGT-A is not available in principle, 2.1PN zygotes are rarely used in the embryo transfer. On the other hand, the size of the micro pronucleus in 2.1PN zygotes has not been clearly defined, and it is difficult to determine differences between 2.1PN and 3PN zygotes. Study design, size, duration The study was performed retrospectively on 2463 cycles of in vitro fertilization (IVF) conducted at our clinic between August 2020 and December 2021. A total of 3073 embryos underwent conventional-IVF (c-IVF) or intracytoplasmic sperm injection (ICSI) and were cultured in the time-lapse incubator, of which 221 zygotes with three pronuclei were used in the study. Participants/materials, setting, methods The diameter of the three PNs at one hour before syngamy from time-lapse images; 2.1 PN and 3PN zygotes were classified in the report by Capalbo et al. (2017). The age of the patients and the method of insemination between the groups were compared, and the diameter of the micro PN was analysed. Moreover, logistic regression analysis was performed to investigate the predictor of 2.1PN zygotes from the morphological characteristics of oocytes at ICSI. Main results and the role of chance The mean age of each patient was 42.9 years for 2.1PN zygotes and 39.8 years for 3PN zygotes, significantly higher for 2.1PN zygotes (P =0.003). On the other hand, when comparing the stage of oocyte maturation at the time of oocyte retrieval, there was no significant difference (P =0.749). According to the insemination method, the incidence of 2.1PN zygotes was significantly higher in ICSI (including rescue-ICSI) compared to c-IVF: 32.9% [95%CI: 22.5-44.6%] vs 2.4% [95%CI: 0.1-12.9%] (P <0.001). In terms of ICSI-derived zygotes, the mean age was also significantly higher for 2.1PN zygotes compared to 3PN zygotes: 43.3 years vs. 40.9 years (P =0.03). The diameter of micro PNs calculated using the receiver operating characteristics (ROC) curve from the measurements of the diameter was less than 15 μm (AUC [95%CI]: AUC=0.988 [0.975-1.00]). Logistic regression analysis using age, position of the oocyte spindle at ICSI, cytoplasmic viscosity, and condition of the cell membrane as explanatory variables revealed a significant difference only in age (P =0.0154, odds ratio [95%CI]: 1.18 [1.03-1.35]) and no statistically significant oocyte morphological characteristics. Limitations, reasons for caution In this study, we have not investigated whether 2.1 PN zygotes become blastocysts. It will be necessary to further examine the criteria for 2.1PN along with chromosome testing to investigate the use of 2.1PN-derived blastocysts. Wider implications of the findings A pronucleus of less than 15 μm in diameter can be considered a micro PN. Compared to 3PN zygotes, 2.1PN zygotes were more frequently observed in older patients and in ICSI-derived zygotes. However, it is difficult to predict the incidence of 2.1PN zygotes from the oocytes’ morphological characteristics at ICSI. Trial registration number not applicable

The aim of this study is to analyse the association between blastocyst diameter and TVs development, and to examine the efficacy of AH. Blastocysts with a diameter of more than 170 μm leads to high incidence of TVs and AH applied from the incidence should be effective. TVs are protrusion of trophectoderm cells often observed in expanding blastocyst stages. TVs can be observed in expanding blastocysts regardless of Intracytoplasmic sperm injection (ICSI) and Conventional-IVF (C-IVF), when the internal pressure of blastocysts increase. The rate of TVs incidence in blastocysts inseminated by ICSI is higher than that by C-IVF, due to penetration of the needle into the zona pellucida. Moreover, it has been reported that TVs may inhibit blastocyst hatching. However, the developmental timing of TVs is still unclear, and there is no study that has analysed the association between blastocyst diameter and the incidence of TVs. 1) Diameters and TVs incidence of blastocysts by ICSI and C-IVF were measured, and the cut-off value and the area under the curve (AUC) of the receiver operating characteristic (ROC) curve were calculated to estimate the timing of TV incidence. 2) We analysed the clinical pregnancy rates of blastocysts with TVs treated by AH compared to those of blastocysts by C-IVF not subjected to AH. This study included 821 transferred frozen blastocysts ranging from March 2018 to November 2019. The embryos were cultured in a dry incubator after insemination by ICSI or C-IVF. Blastocyst freezing conditions were set at day5 to day7 with a diameter of more than 150 μm in inner diameter of zona pellucida, and this was measured before freezing. The ROC curve was performed using EZR statistical analysis software. 1) The incidence of TVs in blastocysts by ICSI and C-IVF was 27.5% (117/424) and 14.6% (58/397) respectively. The rate of the incidence of TVs in blastocysts inseminated by ICSI and C-IVF; 8.6% (12/140) and 0.95% (1/105) in 150–159 μm, 12.7% (14/110) and 8.2% (6/73) in 160–169 μm, 40.6% (28/69) and 10.5% (6/57) in 170–179 μm, 55.6% (30/54) and 25.5% (13/51) in 180–189 μm, 66.7% (20/30) and 35.7% (10/28) in 190–199 μm, and 68.4% (13/19) and 26.8% (22/82) in the diameter of more than 200 μm. The cut-off value of the ROC curve was respectively 170 μm (sensitivity 78.6% and specificity 73.0%) and 176 μm (sensitivity 84.5% and specificity 59.6%) in the diameter; the AUC was 0.8 [95%CI:0.752–0.848] and 0.74 [95%CI:0.687–0.793] respectively. 2) The clinical pregnancy rate of TVs blastocyst vs C-IVF blastocyst was 52.7% (88/167) vs 57.8% (37/64) respectively. There is no significant difference between the two clinical pregnancy rates (P = 0.556). The findings of this study have to be seen in light of some limitations. Since this study aimed to analyse the incidence of TVs based on blastocyst size, we did not take into account the grade according to the Gardner classification and the number of trophectoderm cells. Wider implications of the findings: Blastocysts inseminated by ICSI and C-IVF were highly likely to have TVs above 170 μm and 176 μm respectively. The clinical pregnancy rates of the blastocyst with TV treated by AH was similar to those of the C-IVF blastocyst. Not applicable