The effect of various assisted hatching techniques on the mouse early embryo development

Abstract

ObjectiveIn 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.MethodsFour- 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.ResultsFollowing 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).ConclusionOur 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.

The effect of artificial shrinkage and assisted hatching on the development of mouse blastocysts and cell number after vitrification

“…The rates of hatched blastocyst formation were 49.1% and 40.4% in the qLAH and qLZT-AH groups, respectively, which were significantly higher than in the groups which a smaller area of the ZP was eliminated. Their study was referenced by Park et al [ 25 ], who found a significantly higher rate of formation of hatched blastocysts after 72 hours of culture in the qLAH and qLZT-AH groups than in the control groups. In the present study, the rate of hatched blastocyst embryos was consistently the highest in the AS/qLZT-AH/V-T group ( Table 1 ).…”

“…Cieslak et al [ 24 ] reported that a pregnancy rate of 42.0% was obtained in a group that underwent three-dimensional partial zona dissection, using a micropipette rather than strong acid for dissecting the ZP in preimplantation genetic diagnosis and AH, which was then rotated for another round of dissection, compared to a rate of 33.3% in the control group. Additionally, Park et al [ 25 ] showed that the rate of fully hatched embryos cultivated for 72 hours after AS was 9.0% in the group in which cMAH was performed, compared to 0% in the group in which single mechanical AH. The present experiment was largely based on the report of Khalifa et al [ 26 ], who found a high incidence of hatched blastocyte embryo formation using quarter-LAH (qLAH) and qLZ-AH, which are helpful techniques for encouraging blastocysts to hatch because they eliminate the rather large area of the ZP.…”

Utilization of Assisted Hatching Techniques to Enhance Embryo Implantation

“…Teknik ini kemudian banyak dikembangkan melalui penelitian untuk mengoptimalkan hasil, antara lain dengan cross Mechanical Assisted Hatching (cMAH). Metode yang dikembangkan oleh Park et al (2014) sebenarnya adalah pengembangan dari PZD. Namun, cMAH dikerjakan dengan membuat dua kali sayatan yang saling bersilangan.…”

“…Berbagai pengembangan metode AH menggunakan sinar laser terus dilakukan. Salah satunya adalah beberapa pengembangan penggunaan berbagai jenis sinar laser seperti 1,48 µm infrared laser diode (Park et al 2014), ZILOS-TK infrared laser optical system (Makrakis et al 2006), OCTAX laser shot system (Nishio et al 2006) dan erbium-yttriumaluminium-garnet (Er:YAG) (Obruca et al 1997).…”

“…Jenis laser non-kontak yang menggunakan infrared dioda dengan panjang gelombang 1,48 µm dapat menghasilkan luasan area tembakan yang besar dan dapat dikerjakan hanya dalam waktu 10 detik (Park et al 2014 (Blake et al 2001). Jenis sinar laser lain yaitu Er:YAG dengan diameter tip laser fibre 20 µm, energi yang digunakan sebesar 10 µJ dan kedalaman penetrasi 3 µm menjadi salah satu cara untuk melakukan AH.…”