Morphokinetic Behavior of the Second Polar Body in Human Zygotes as a Predictor for Embryonic Developmental Potential: An Exploratory Study Based on Time-Lapse Observation

Abstract

Time-lapse imaging has made possible the detailed observation of all stages of embryonic development, including also from the extrusion of the second polar body up to the first cleavage. By extensive observation, we achieved detection of a variety of behaviors of PBIIs such as (a) morphologically static behavior, (b) amoeboid movement, (c) shrinking, (d) fragmenting, and (e) ruffling. Retrospective analysis was performed on 282 ICSI zygotes derived from 69 ART treatment cycles from January to August 2019. Zygotes with morphologically static PBIIs (a) and PBIIs showing various behaviors (b)~(e) were classified into Group 1 (n = 70) and Group 2 (n = 212), respectively. Based on the rates of irregular division, good quality embryos, and the time from the PBII extrusion, pronuclear breakdown to the first cleavage was compared between groups (Study 1). Furthermore, the relationship between the type of PBII behaviors and ploidy in 94 biopsied blastocysts from 15 cycles was examined, in which one or more euploid embryos were obtained between August 2021 and July 2024 (Study 2). The results showed that good quality embryos tended to have morphologically static PBIIs, and that euploid embryos were absent in embryos with fragmenting and ruffling PBIIs. The behavior of PBIIs may be a new predictor of embryonic developmental potential, and, in the future, morphokinetic behaviors of PBIIs may be a useful parameter for AI-assisted embryo evaluation systems.

Keywords: ART; embryo development; morphokinetic behavior; second polar body; time-lapse cinematography.

Figure 1

Schematic diagram of various behaviors of the PBII.

Figure 2

The sequential still images of zygotes from the time of second polar body (PBII) extrusion to the first cleavage. (A) Static: PBII rarely moved and its shape did not change from the time of extrusion to the first cleavage. (B) Amoeboid: PBII moved and changed its shape like amoeba after the extrusion to the first cleavage. (C) Shrinking: The size of PBII just before the first cleavage was smaller than immediately after the extrusion. (D) Fragmenting: A round-shaped PBII gradually fragmented over time. (E) Ruffling: The shape of PBII ruffled during the observation. White arrowheads indicate the PBII. Magnification:.

Figure 3

The developmental events before the first cleavage with time interval values. Although there was no significant difference in the time interval from PBII extrusion up to the first cleavage between the two groups, the time interval from PNBD to the FC was significantly longer for Group 2 than for Group 1. Data are presented as mean ± SD in hours. * Statistical significance: p < 0.05. FC: first cleavage; PBII: second polar body; PNBD: pronuclear breakdown.

Figure 4

(a) PGT-A results for each PBII morphokinetic behavior. Embryos with static PBIIs: 4 euploid embryos, 2 embryos with low-level mosaicism, 5 embryos with high-level mosaicism, and 10 aneuploid embryos. Embryos with amoeboid PBIIs: 19 euploid embryos, 2 embryos with low-level mosaicism, 2 embryos with high-level mosaicism, and 15 aneuploid embryos. Embryos with shrinking PBIIs: 8 euploid embryos, 2 embryos with low-level mosaicism, and 3 aneuploid embryos. Embryos with fragmenting PBIIs: 1 with low-level mosaicism and 7 aneuploid embryos. Embryos with ruffling PBIIs: 3 aneuploid embryos. (b) The number of euploid embryos in each group based on the PBII behaviors. There were 31 euploid embryos with PBIIs showing static, amoebic, and shrinking behaviors (37%; n = 31/72), whereas there were no euploid embryos with ruffling and fragmenting PBIIs (0%; n = 0/11). (* p = 0.00126, ^† p = 0.00596). The numbers of embryos are shown in the bars. PBII: second polar body.

Figure 4

(a) PGT-A results for each PBII morphokinetic behavior. Embryos with static PBIIs: 4 euploid embryos, 2 embryos with low-level mosaicism, 5 embryos with high-level mosaicism, and 10 aneuploid embryos. Embryos with amoeboid PBIIs: 19 euploid embryos, 2 embryos with low-level mosaicism, 2 embryos with high-level mosaicism, and 15 aneuploid embryos. Embryos with shrinking PBIIs: 8 euploid embryos, 2 embryos with low-level mosaicism, and 3 aneuploid embryos. Embryos with fragmenting PBIIs: 1 with low-level mosaicism and 7 aneuploid embryos. Embryos with ruffling PBIIs: 3 aneuploid embryos. (b) The number of euploid embryos in each group based on the PBII behaviors. There were 31 euploid embryos with PBIIs showing static, amoebic, and shrinking behaviors (37%; n = 31/72), whereas there were no euploid embryos with ruffling and fragmenting PBIIs (0%; n = 0/11). (* p = 0.00126, ^† p = 0.00596). The numbers of embryos are shown in the bars. PBII: second polar body.

Figure 5

(a) Participants and setting of Study 1. In total, 306 normally fertilized ICSI-zygotes were obtained. Of those, 24 were excluded from the study due to difficulties in observing the PBII. A total of 282 zygotes were included in the study. These 282 zygotes were allocated into two groups according to the morphokinetic behavior of the PBII. In Group 1 zygotes with static PBII were included (n = 70) and in Group 2 the remaining 4 types of PB II were included (n = 212). ICSI: intracytoplasmic sperm injection; PBII: second polar body. (b) Subjects and setting of Study 2. Ninety-four blastocysts that had undergone biopsies were selected for analysis. Of those, nine were excluded due to difficulties in identifying the PBII and another two due to no results. A total of 83 blastocysts were included for analysis from the zygote stage up to becoming a blastocyst suitable for biopsy. The 83 zygotes were allocated into five categories according to the behaviors of the PBII (static, amoeboid, shrinking, fragmenting, and ruffling). The blastocysts after the PGT results were allocated as euploid, low-level mosaic, high-level mosaic, and aneuploidy.