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The location of “ 8 ” -shaped hatching influences inner cell mass formation in mouse blastocysts

View Article: PubMed Central - PubMed

ABSTRACT

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed “8”-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during “8”-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify “8”-shaped hatching, and we classified the “8”-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were “8”-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the “8”-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida―the portion defined as the “outside blastocyst”―after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in “8”-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of “8”-shaped hatching behaviors could yield indices for accurately evaluating embryo quality.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0175150.g001: 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.

Mentions: Embryos were imaged using a TLM system (Primo Vision, Vitrolife) at 5-min intervals, and were examined until 120 h post-insemination based on referring to previous work [5]. The embryos were continuously evaluated and the collected TLM data were used for sorting the embryos according to the hatching mode (Fig 1A); as per the criteria reported in previous work [5], the hatching modes were differentiated as follows: When blastocysts featured 2 blastocoels divided by the ZP and the ZP-opening size was ≤25 μm, they were regarded as “8”-shaped-hatching blastocysts (Fig 1B(a-d)). Conversely, when the opening size increased to >25 μm during the monitoring period, or when the newly generated openings of the blastocysts were >25 μm in diameter, the blastocysts were regarded as U-shaped-hatching blastocysts (Fig 1B(e)). Embryos harboring multiple small-diameter hatching points (Fig 1B(f)) were excluded from this study.


The location of “ 8 ” -shaped hatching influences inner cell mass formation in mouse blastocysts
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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383253&req=5

pone.0175150.g001: 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.
Mentions: Embryos were imaged using a TLM system (Primo Vision, Vitrolife) at 5-min intervals, and were examined until 120 h post-insemination based on referring to previous work [5]. The embryos were continuously evaluated and the collected TLM data were used for sorting the embryos according to the hatching mode (Fig 1A); as per the criteria reported in previous work [5], the hatching modes were differentiated as follows: When blastocysts featured 2 blastocoels divided by the ZP and the ZP-opening size was ≤25 μm, they were regarded as “8”-shaped-hatching blastocysts (Fig 1B(a-d)). Conversely, when the opening size increased to >25 μm during the monitoring period, or when the newly generated openings of the blastocysts were >25 μm in diameter, the blastocysts were regarded as U-shaped-hatching blastocysts (Fig 1B(e)). Embryos harboring multiple small-diameter hatching points (Fig 1B(f)) were excluded from this study.

View Article: PubMed Central - PubMed

ABSTRACT

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed “8”-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during “8”-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify “8”-shaped hatching, and we classified the “8”-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were “8”-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the “8”-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida―the portion defined as the “outside blastocyst”―after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in “8”-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of “8”-shaped hatching behaviors could yield indices for accurately evaluating embryo quality.

No MeSH data available.


Related in: MedlinePlus