Fig 1.
Sorting and evaluation of blastocysts.
Flowchart (A) and schematic of the morphology at 120 h post-insemination (B) of the 104 blastocysts obtained in IVF. Based on morphology, the embryos can be classified into 8 types (B, a-h). Hatching embryos, based on their morphology, can be classified into “8”-shaped-hatching (a-d), U-shaped-hatching (e), and multiple-hatching-site (f) blastocysts. Only 9 of the U-shaped-hatching blastocysts hatched completely (g). The “8”-shaped-hatching blastocysts were classified according to the relationship between the hatching site and inner cell mass (ICM) position into the Near group (a-c) and Far group (d). The Near-group embryos were further classified into embryos in which the ICM was located both inside the zona pellucida (ZP) and in the outside blastocyst (b); embryos in which all of the ICM was included inside the ZP (a); and embryos in which all of the ICM was included inside the outside blastocyst (c). The term “outside blastocyst” refers exclusively to the specific portion of the blastocyst that lies outside the ZP.
Fig 2.
The outside blastocyst immediately before (A) and immediately after (B) collapse was set in an ellipse and the lengths of the long and short axes were measured in order to calculate the cross-sectional area. An area reduction of ≥50% was defined as a collapse.
Fig 3.
Method used for determining ICM size.
Oct3/4 (red) was stained with specific antibodies, and Oct3/4-positive cell masses were identified as ICM. The 3D confocal image of each blastocyst was rotated to set the ICM center on the cross-section (A, B), and then the ICM end-to-end distance along the curved surface (C) was measured by following each of the perpendicular lines (curves) on the blastocyst image. Subsequently, these measurements for each blastocyst were averaged to obtain the “width” as an indicator of the size of the ICM in the blastocyst. Scale bar, 20 μm.
Fig 4.
Classification of Near and Far groups.
The ICM center (*) was identified and blastocysts were divided into two halves as demarcated in the figure, with the hatching site set as a vertex. Embryos with the hatching sites on the nearer side and the farther side from the ICM center were sorted into the “Near group” and “Far group,” respectively. The embryo shown in this figure was sorted into the Far group.
Fig 5.
Immunostaining of “8”-shaped-hatching and U-shaped-hatching blastocysts.
Cdx2 (green) and Oct3/4 (red) were stained with specific antibodies, and nuclei were stained with Hoechst (blue). In the case of “8”-shaped hatching, the figure shows embryos in the Near group (A–D) and Far group (E–H). Whereas the ICM tended to be morphologically compact in the Far group (G), it was scattered in the Near group (C; see Fig 7C).
Fig 6.
Time-lapse and immunostaining images of a U-shaped-hatching blastocyst that hatched completely, in which an initial “8”-shaped hatching occurred near the ICM.
(A) At 90 h 45 min after insemination. Near the ICM (surrounded by arrowheads), an “8”-shaped hatch appears (arrow). (B) At 117 h 57 min after insemination. U-shaped hatching appears (arrow) in this embryo. (C) At 119 h 47 min after insemination. The embryo has hatched completely. (D) Immunostaining at 120 h post-insemination. The result confirmed that the ICM, stained by anti-Oct3/4 (red), is scattered. Scale bar, 20 μm.
Table 1.
Rate and median of inside/outside collapse in “8”-shaped-hatching, U-shaped-hatching, and unhatched blastocysts.
Fig 7.
ICM size and cell number at 120 h post-insemination, sorted according to type of developmental behavior of blastocysts: “8”-shaped hatching and U-shaped hatching, hatching position relative to the ICM, and occurrence of outside collapse.
(A, B) Comparison by hatching mode. (C, D) Comparison by hatching position. The ICM was significantly wider in the Near group than in the Far group (*P = 0.0091, Welch’s t test). (E, F) Comparison based on occurrence of outside collapse.
Fig 8.
Time-lapse and immunostaining images of “8”-shaped hatching.
(A) At 119 h 30 min post-insemination. The outside blastocyst is expanded. (B) At 119 h 35 min post-insemination. The outside blastocyst has collapsed (arrow). (C) At 119 h 59 min post-insemination. The outside blastocyst has re-expanded. (D) Immunostaining at 120 h post-insemination, immediately after the time point shown in C. A merged image of Oct3/4 (red), Cdx2 (green), and Hoechst (blue) staining is shown. Oct3/4-positive cells (arrowhead) were attached to the TE of the outside blastocyst opposite the hatching site. Scale bar, 20 μm.
Fig 9.
Immunostaining of an “8”-shaped-hatching blastocyst at 120 h post-insemination.
The images show ZO2 (red) and Na+/K+-ATPase (green) staining, nuclei stained with Hoechst (blue), and the midplane section (A–D) and maximum intensity projection (E–H). The two TE-structure proteins Na+/K+-ATPase and ZO2 were similarly expressed inside and outside the ZP. Scale bar, 20 μm.