Fig 1.
Measurement of the blastocyst cross-sectional area using TLC.
The blastocyst cross-section is prominently elliptical (area outlined in yellow); the major axis (red) and minor axis (blue) were measured using ‘Primo Vision analyzer’ drawing tools. (a) Fully expanded blastocyst immediately before contraction. (b) Timepoint at which the cross-sectional area of the contracted blastocyst was the smallest.
Fig 2.
Definition of blastocyst contraction using TLC.
(a) Original image of contracted blastocyst. (b) Surface of the contracted blastocyst analyzed using ‘Primo Vision analyzer’ drawing tools; the inner surface of the zona pellucida and outer surface of the TE are outlined in yellow. Contraction is defined as failure of these lines to coincide; in this image, the cells are 70% separated.
Fig 3.
Number of ICM and TE cells counted using IMARIS imaging analysis software.
(a) A 3D projection of an immunostained human expanded blastocyst (original image). (b) Number of ICM cells; red-stained nuclei were automatically marked with white spheres and counted. (c) Number of TE cells; green-stained nuclei were automatically marked and counted. Oct4 (red), Cdx2 (green), and chromatin (blue) nuclear staining. Scale bar, 70 μm.
Table 1.
Characteristics of Expanded and Non-Expanded blastocysts.
Fig 4.
Presence patterns of Oct4 and Cdx2 in an immunostained human blastocyst.
The immunostained samples were observed using confocal microscopy. (a) Blastocyst in the Expanded group; only Oct4-positive nuclei were found in the ICM and only Cdx2-positive nuclei were found in the TE. (b) Blastocyst in the Expanded group; double-positive (Oct4- and Cdx2-positive) nuclei were found in the ICM and TE regions. (c) Blastocyst in the Non-Expanded group; most of nuclei were double-positive (Oct4- and Cdx2-positive). Oct4 (red), Cdx2 (green), and chromatin (blue) nuclear staining.
Fig 5.
3D projection of an immunostained human expanded blastocyst observed from various directions.
(a) Images of uniformly distributed TE cells. Many sperm heads are attached to zona pellucida. In the case of IVF, sperm heads may remain. (b) Images of unevenly distributed TE cells. Oct4 (red), Cdx2 (green), and chromatin (blue) nuclear staining.
Fig 6.
Number of ICM and TE cells in the Expanded (n = 28) and Non-Expanded groups (n = 10).
(a) The number of ICM cells is significantly higher in the Expanded group than in the Non-Expanded group (*p = 0.0162, Mann-Whitney U test). Horizontal bar shows the mean. (b) The number of TE cells is significantly higher in the Expanded group than in the Non-Expanded group (**p < 0.0001, Mann-Whitney U test). Horizontal bar shows the mean.
Fig 7.
Correlation between the initial expansion rate and cell numbers.
(a) Initial expansion rate and the number of ICM cells. (b) Initial expansion rate and the number of TE cells. Squares represent blastocysts that started hatching in culture within 22 h. Black circles represent those that did not start hatching.
Fig 8.
Correlation between the number of TE cells per unit of blastocyst maximum cross-sectional area and the number of contractions.
Squares represent blastocysts that started hatching in culture within 22 h. Black circles represent those that did not start hatching.
Fig 9.
Representative images of human blastocysts with high (a) or low (b) numbers of TE cells per maximum expansion cross-sectional area observed using confocal microscopy.
(a) Blastocyst fixed in hatching stage. (b) Blastocyst fixed in expanded stage. Oct4 (red), Cdx2 (green), and chromatin (blue) nuclear staining. Scale bar, 100 μm.
Table 2.
Correlation between the number of ICM and TE cells, initial expansion rate, and hatching with maternal age (< 35 or ≥ 35).
Table 3.
Correlations between the dpf and the numbers of ICM and TE cells, initial expansion rate, and hatching.
Table 4.
Correlation between the number of contractions in each embryo and the number of TE cells per maximum expansion cross-sectional area with age (< 35 or ≥ 35) and dpf (5 or 6).