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. 2014 Jun;41(2):68-74.
doi: 10.5653/cerm.2014.41.2.68. Epub 2014 Jun 30.

The Effect of Various Assisted Hatching Techniques on the Mouse Early Embryo Development

Free PMC article

The Effect of Various Assisted Hatching Techniques on the Mouse Early Embryo Development

Sung Baek Park et al. Clin Exp Reprod Med. .
Free PMC article


Objective: In search of an ideal method of assisted hatching (AH), we compared the effects of conventional micropipette-AH and laser-AH on the blastocyst formation rate (BFR) and blastocyst cell numbers.

Methods: Four- to five-week-old ICR female mice were paired with male mice after superovulation using Pregnant mare's serum gonadotropin (PMSG) and hCG. The two-cell embryos were flushed from the oviducts of female mice. The retrieved two-cell embryos underwent one of five AH procedures: single mechanical assisted hatching (sMAH); cross mechanical assisted hatching (cMAH); single laser assisted hatching (sLAH); quarter laser assisted hatching (qLAH); and quarter laser zona thinning assisted hatching (qLZT-AH). After 72 hours incubation, double immunofluorescence staining was performed.

Results: Following a 72 hours incubation, a higher hatching BFR was observed in the control, sMAH, cMAH, and sLAH groups, compared to those in the qLAH and qLZT-AH groups (p<0.05). The hatched BFR was significantly higher in the qLAH and qLZT-AH groups than in the others (p<0.05 for each group). The inner cell mass (ICM) was higher in the control and sMAH group (p<0.05). The trophectoderm cell number was higher in the cMAH and qLAH groups (p<0.05).

Conclusion: Our results showed that the hatched BFR was higher in groups exposed the the qLAH and qLZT-AH methods compared to groups exposed to other AH methods. In the qLAH group, although the total cell number was significantly higher than in controls, the ICM ratio was significantly lower in than controls.

Keywords: Assisted hatching; Cell number; Laser assisted hatching; Mechanical assisted hatching; Mouse embryo.

Conflict of interest statement

No potential conflict of interest relevant to this article was reported.


Figure 1
Figure 1
Comparison images of naturally-hatching and assisted hatching blastocysts, and differentially stained features of blastocyst in mouse (the scale bars are 50 µm, ×400). (A) Naturally hatching blastocyst (i.e., without assisted hatching). (B) Hatching blastocyst, with assisted hatching. (C) Differentially stained hatching blastocyst (inner cell mass, blue; trophectoderm, pink).
Figure 2
Figure 2
Schematic drawing of types of assisted hatching techniques. (A) Single mechanical assisted hatching. (B) Cross mechanical assisted hatching. (C) Single laser assisted hatching. (D) Quarter laser assisted hatching. (E) Quarter laser zona thinning-assisted hatching.
Figure 3
Figure 3
Photographs of techniques for sMAH and cMAH (the scale bars are 20 µm, ×400). (A) Mouse two-cell embryo fixed by holding pipette. (B) Entry position of the first cut for sMAH. (C) Embryos to rub holding pipette. (D) Mechanical assisted hatching. Arrow head, slit opening after sMAH. (E) Vertical rotation (curved arrow) of the embryo after the first slit is made and entry position of the second cut for the cross-shaped opening. (F) Mechanical assisted hatching. Arrow, slit opening after cMAH. sMAH, single mechanical assisted hatching; cMAH, cross mechanical assisted hatching.

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