Effects of laser-assisted hatching and exposure time to vitrification solution on mouse embryo development / 대한생식의학회지

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±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±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±5.1, p < 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group (73.2±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.

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

OBJECTIVE: The goal of this study was to ascertain optimal assisted hatching (AH) method in frozen embryo transfer. We compared the effect of depending on whether mechanical or laser-AH was performed before or after the vitrification of embryo development rate and blastocyst cell numbers. METHODS: In order to induce superovulation, pregnant mare's serum gonadotropin followed by human chorionic gonadotropin were injected into 4- to 5-week-old female mice. 2-cell embryos were then collected by flushing out the oviducts. The Expanded blastocysts were recovered after the collected embryos were incubated for 48 hours, and were then subjected to artificial shrinkage (AS) and cross-mechanical AH (cMAH) or quarter-laser zona thinning-AH (qLZT-AH) were carried out using the expanded blastocysts before or after vitrification. After 48 hours of incubation, followed by vitrification and thawing (V-T), and blastocysts were fluorescence stained and observed. RESULTS: The rate of formation of hatched blastocysts after 24 and 72 hours of incubation was significantly higher in the AS/qLZT-AH/V-T group than in the other groups (p<0.05). The cell number of the inner cell mass was higher in AS/V-T/non-AH and AS/V-T/cMAH groups than those of others (p<0.05). In the control group, the number of trophectoderm and the total cell number were higher than in the AS-AH group (p<0.05). CONCLUSION: The above results suggest that AS and AH in vitrification of expanded blastocysts lead to the more efficient formation of hatched blastocysts in mice.

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

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.

Effects of Glucose on Blastocyst Formation and Their Cell Numbers of Mouse Embryos / 대한산부인과학회잡지

OBJECTIVE: Mammalian embryos undergo changes of energy environment for transfer from oviduct to uterus. An experimental design was used to examine the effects of glucose on the development in vitro of mouse embryos. METHODS: Two cell embryos were recovered from ICR female mice (3-4 weeks) at 46-50 hrs after 5 IU hCG injection (mated just after hCG injection) and cultured in 50 micro gram MEM droplets supplemented with nothing (control; n=46), 0.5 mM glucose (Group A; n=46) or 3.15 mM glucose (Group B; n=46) under mineral oil. All experimental media were supplemented with 20% human follicular fluid. Results were observed: (i) the number of zona-intact blastocysts (ZiB); (ii) the number of zona-escaped blastocysts (ZeB; hatching~hatched); (iii) the mean cell numbers; and (iv) the proportion of inncer cell mass (% ICM) in the blastocysts. RESULTS: Total blastocyst formation rates were (NS) in glucose groups (group A: 52.2; B: 47.8%) than control group (60.9%). ZiB rates the highest (p<0.05) in control (47.8%) than those in group A (21.7%) and B (28.3%). ZeB rates the highest (NS) in group A (30.4%) than those in control (13.0%) and group B (19.6%). Blastocysts, cultured in group B (50.5), had the highest (NS) mean cell numbers compared with the others (control: 39.2; group A: 45.6). The % ICM in blastocysts cultured in group A (20.6%) was the highest (NS) than those of other tested groups (control: 15.2%; group B: 13.9%). CONCLUSION: This study shows that a low dose of glucose added to culture medium increases the developmental capacity of 2 cell embryos in mice.

Effects of Exposure of Propidium Iodide and Bisbenzimide on Differential Staining of Mouse Blastocysts / 대한불임학회지

OBJECTIVE: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. MATERIALS AND METHODS: A total 964 blastocysts (early~hatched) was exposed to PI (n=831) (group I: