• Clinical and Experimental Reproductive Medicine
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• v.41 no.2
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• pp.68-74
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• 2014

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.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.147-153
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• 2001

Objective: The present study was performed to investigate the efficiency of partial laser assisted hatching (p-LAH; lased 1/2 ZP width from ZP edge) on hatching of mouse blastocysts. Methods: We used non-contact $1.48{\mu}m$ diode laser (MTM, Switzland) to create a precise hole on zona pellucida. 2-cell embryos were collected from the mouse (ICR) oviduct at 48 hours after hCG administration. Collected 2-cell embryos were cultured in the P-1 medium supplemented with 0.4% BSA. For experiments, embryos at 8-cell stage were used after $20{\sim}22$ hours in culture. After conventional (c-LAH) or partial laser assisted hatching, the embryos were further cultured in P-1 medium supplemented with 0.4% BSA for 3 days. To compare efficiency of complete and partial laser assisted hatching, hatching rate, hatching time and blastocyst diameter and zona pellucida thickness at hatching time were investigated. Embryos were examined every 12 hours. Blastocyst diameter and zona pellucida thickness at hatching time were measured with an ocular micrometer. Results: Hatching rates of p-LAH group (84.2%) was significantly higher than that of control group (39.3%), but there was no difference between the p-LAH (84.2%) and c-LAH (91.2%). p-LAH group was hatched 12 hours earlier than control group, but hatched 12 hours later than c-LAH group. The diameter of blastocyst at hatching time of p-LAH group ($113.1{\pm}6.4{\mu}m$) was smaller than that of control group ($122.2{\pm}5.0{\mu}m$), but larger than that of c-LAH group ($102.2{\pm}2.7{\mu}m$). Zona pellucida thickness at hatching time of p-LAH group ($6.4{\pm}0.9{\mu}m$) was thicker than that of control group ($4.5{\pm}1.5{\mu}m$), but thinner than that of c-LAH group ($10.0{\pm}0.8{\mu}m$). Conclusion: These results suggest that p-LAH may maintains the cell arrangement of early embryos to ensure successful development and prevent precocious hatching of blastocyst when compare to c-LAH and conventional (acidic tyrode) AH. Thus, p-LAH may provide a valuable and effective AH technique for human ART program.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.2
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• pp.51-57
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• 2015

Objective: This study aimed to examine the effect of laser-assisted zona thinning (LAZT) at one or four-points on the blastocyst formation and hatching process in mice with respect to female age. Methods: Eight-cell or morula embryos collected from superovulated C57BL female mice with different ages (6-11 and 28-31 weeks) were treated with LAZT at one-point (LAZT1) or four-points (LAZT4). The zona pellucida was thinned to more than 70% of its initial thickness by making two holes of $15-20{\mu}m$. Results: In the young mice, LAZT resulted in a significant increase in early hatching and hatching rates compared to the control group (p<0.05). However, in the old mice, LAZT significantly increased blastocyst formation as well as early hatching and hatching compared to the controls (p<0.05). These effects were more remarkable in LAZT4 than in LAZT1 and in aged mice than in young ones. Conclusion: These results show that multi-point LAZT leads to a significant improvement of blastocyst formation and hatching in mice compared to controls.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.1-4
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• 2008

This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze $2{\sim}4$ cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in $LN_2$. Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for $96{\sim}120\;hr$ and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.185-192
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• 1999

The present study was performed to investigate the efficacy and safety of laser assisted hatching (AH) on mouse embryos. Non-contact $1.48{\mu}m$ diode laser system used to create a precise hole on zona pellucida. 2-cell embryos were collected from the mice (ICR) that had the coitus vaginal plug confirmed at 48 hours after hCG injection. Collected 2-cell embryos were cultured in the HTF medium supplemented with 0.4% BSA. For experiments, embryos at 8-cell stage were used after 18-22 hours in culture. After assisted hatching, the embryos were further cultured in HTF medium containing 0.1% PVP (anti-hatching system) for 3 days. For evaluate efficiency of laser on mouse embryo hatching, the effect of AH methods (acidic tyrode, pronase and laser), the number of artificial holes (1, 2 and 3 hole) and the irradiation time of laser (2, 4, 6, 8 and 10 ms) were examined. Hatching rates of laser AH group (95.2%) was significantly higher than that of control group (50.8%), but there was no differences among the laser (95.2%), acidic tyrode (100%) and pronase (98.5%) groups. Hatching rates of the number of zona pellucida opening by laser, there were no differences among the 1 hole (87.5%),2 hole (92.1%) and 3 hole (85.9%) groups. Developmental and hatching rates of embryos according to laser irradiation time were similar in the treatment groups. Therefore, these results suggest that laser AH using non-contact $1.48{\mu}m$ diode laser is a simple and accurate and effective procedure for AH. Based on these results, laser AH could be use safely for human ART program.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.123-128
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• 1998

Implantation rates remain low following human in vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). In this study, assisted hatching (AH) by zona drilling using acidic Tyrode's solution was performed in 320 patients, due to their age of more than 38 years (group A), the thick zona pellucida (group Z; $ZP\geq0.18{\mu}m$), and failures in implantation more than 3 times in previous IVF-ET trial (group P). This study was designed firstly, to study the effects of AH on the outcomes of IVF-ET according to the indications and secondly, to verify the appropriate application of AH. The results were as follows; 1. There was no difference in pregnancy rate between AH group (26.6%) and non-AH group (26.5%). 2. Assisted hatching (AH) showed significantly higher pregnancy rate of the patients with thick zona pellucid a than those of the patients with age factor and with the history of repeated implantation failure. But in the patients with age factor only, AH resulted in higher pregnancy rate. 3. Interestingly, the patients with complex factors including zona factor (Z: 33.9%; ZA: 30.4%; ZP: 31.6%; ZAP: 21.4%) showed higher pregnancy rates than other complex factors excluding zona factor (A: 24.4%, P: 0%; AP: 10.8%). From these results, AH is more helpful to the patients with thick zona pellucida rather than patients with older age and/or previous repeated implantation failure.

• 대한생식의학회:학술대회논문집
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• 2000.02a
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• pp.267-276
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• 2000

• Clinical and Experimental Reproductive Medicine
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• v.44 no.4
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• pp.193-200
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• 2017

Objective: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. Methods: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. Results: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p< 0.05). In the control-8 group ($22.1{\pm}4.6$), the cell number of the inner cell mass was higher than in the LAH groups (p< 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group ($92.8{\pm}8.9$) than in the others (p< 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group ($19.5{\pm}5.1$, p< 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group ($73.2{\pm}12.1$) than in the other groups, but without statistical significance. Conclusion: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.275-283
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• 2008

Objective: This study was conducted to investigate the effects of the artificial shrinkage and assisted hatching (PZD; patial zona dissetion) before vitrification on the development of vitrified mouse expanding blastocyst. Methods: Mouse 2-cell embryos were collected and cultured in G1.1 and G2.2 to expanding blastocyst. For artificial shrinkage (AS) the micro injection pipette was inserted into blastocoele cavity and blastocoele fluid was aspirated. For assisted hatching (AH) PZD method was used. Control group was -AS/-AH and treatment groups were -AS/+AH, +AS/-AH and +AS/+AH. After AS and AH mouse blastocysts were equillibrated in G10 and G10E20 for 3 mins, respectively, and vitrified in G25E25 after loading on capped pulled-straw. Vitrified mouse blastocysts were thawed and cultured for 24 hrs. The survival and hatching rate was compared among one control and three treatment groups. Results: The survival rates were 99%, 92% in +AS/+AH and +AS/-AH groups and 54%, 58% in -AS/-AH and -AS/+AH group, respectively. The survival rate was significantly higher in +AS group than in -AS group (p<0.01). Hatching rates were 34%, 96% in -AS/-AH and -AS/+AH groups and 41%, 100% in +AS/-AH and +AS/+AH, respectively. The hatching rates was higher in +AH group than in -AH group (p<0.01). After thawing recovery rates were 100%. Loading on capped pulled-straw, that is effective and useful method on vitrification. Conclusion: This study showed that artificial shrinkage of blastocoele cavity and assisted hatching (PZD) significantly improved the development of the vitrified mouse expanding blastocysts.

• 대한생식의학회:학술대회논문집
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• 2006.06a
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• pp.155.2-155.2
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• 2006