• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.188-194
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• 1989

These experiments were carried out to investigate fertilizable and developmental ability after zona drilling the unfertilized eggs and the eggs not fertilized by the 1st insemination. The results of in vitro fertilization of the mouse eggs treated by using micromanipulation and acid tyrode's solution with capacitated epididymal spermatozoa were as follows. In the case of ovulated unfertilized eggs, according to sperm count(106, 105, 104 and 103/ml) the rates of in vitro fertlilization treated by zona drilling were 86.0%, 82.0%, 70.0% and 54.0%, respectively, and those of control were 58.0%, 52.0%, 12.0% and 8.0%, respectively. The rates of in vitro fertilization of zona drilled eggs were significantly high compared with those of control, and there were no significant difference between two groups. According to the sperm count the zona drilled eggs developed to the blastocysts were 51.4%, 40.5%, 23.3% and 17.4% and those of control were 35.7%, 26.3%, 0% and 0%, respectively. Also, in the eggs not fertilized by 1st insemination, the fertilization rates of oocytes reinseminated after zona drilling was significantly higher(83.5%) than that of control(34.7%), and the rates of polyspermy were similar. The rates of development to the blastocysts was 18.6% in the zona drilling treated eggs, and that of control was 27.3%, there was no significant difference between two groups. These results indicated that oocytes not fertilized by 1st insemination as well as ovulated unfertilized eggs could be fertilized, improve fertilizing rates by zona drilling treatment, and development potential were normal.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.3
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• pp.118-124
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• 2012

Objective: To review the outcomes of preimplantation genetic diagnosis (PGD) using zona drilling with acid Tyrode's solution (chemical zona pellucida drilling, chemical ZD) and those of partial zona dissection (PZD). Methods: Clinical outcomes of seventy-one couples undergoing 85 PGD cycles from January 2005 to December 2010 were included. Blastocyst formation and the hatching rate, clinical pregnancy rate, ongoing pregnancy rate, implantation rate, and fetal gender ratio of the PZD and chemical ZD groups were compared. Results: Application of PZD resulted in a significantly higher rate of clinical pregnancy (40.7% vs. 15.4%, p=0.022), ongoing pregnancy (35.6% vs. 11.5%, p=0.023), and implantation (18.1% vs. 5.7%, p=0.007) compared with chemical ZD. Among non-transferred embryos, the rate of blastocyst formation on day 5 (49.1% vs. 39.5%, p=0.016) and hatching on day 6 (47.2% vs. 26.5%, p<0.001) were also significantly higher in the PZD group. Conclusion: The mechanical zona dissection (ZD) method showed better outcomes than chemical ZD in terms of the blastocyst development and pregnancy rate. In this study, the fact that chemical ZD was conducted in different period from mechanical method should be considered in interpreting the result.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.193-201
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• 2001

This study was designed to examine the factors affecting in fertilization and development of embryos in vitro, and to examine whether zone drilling by laser irradiation can improve the hatching rate of IVF embryos from DNA marker-proved Hanwoo. DNA markers related to marbling score were identified using DNA fingerprinting with Ml3 probe and restriction enzyme Hae III. Oocytes were aspirated from immature ovarian follicles using a combined method of rectal ovarian-palpation and transvaginal ultrasound-guidance(6.5MHz) under local anesthesia. The aspirated oocytes were washed twice with fresh D-PBS containing 5% FBS and were rewashed 4 to 5 times with TCM-199 containing 5% FBS. A morphological grade of I to IV was assigned to each oocyte. Data were analyzed using the GLM procedure of SAS. Sperm separation methods did not have any significant effect on cleavage or developmental abilities of IVF embryos. Significantly(P<0.05) higher cleavage rate was observed in embryos from GI(60.0%, 3/5), GII(69.2%, 18/26) and GIII(62.1%, 59/95) compared to embryos from GIV oocytes(36.2%, 25/69). And the developmental rate to blastocyst stage was higher(P<0.05) in embryos from GI(33.3%, 1/3) and GII oocytes(38.9%, 7/18) than those from GIII(16.9%,10/59) and GIV oocytes(4.0%, 1/25). There was no significant difference in development of IVF embryos to blastocyst by media for in vitro culture. Proportion of hatched blastocyst was significantly(P<0.05) higher in embryos received zona drilling by laser than those of non-drilled.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.129-134
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• 2018

Objective: In frozen and thawed embryos, the zona pellucida (ZP) can be damaged due to hardening. Laser-assisted hatching (LAH) of embryos can increase the pregnancy rate. This study compared thinning and drilling of the ZP before frozen embryo transfer (FET). Methods: Patients were randomly allocated into two groups for LAH using thinning or drilling on day 2 after thawing. Twenty-five percent of the ZP circumference and 50% of the ZP thickness was removed in the thinning group, and a hole $40{\mu}m$ in diameter was made in the drilling group. Results: A total of 171 in vitro fertilization/intracytoplasmic sperm injection FET cycles, including 85 cycles with drilling LAH and 86 cycles with thinning LAH, were carried out. The thinning group had a similar ${\beta}$-human chorionic gonadotropin-positive rate (38.4% vs. 29.4%), implantation rate (16.5% vs. 14.4%), clinical pregnancy rate (36.0% vs. 25.9%), miscarriage rate (5.8% vs. 2.4%), ongoing pregnancy rate (30.2% vs. 23.5%), and multiple pregnancy rate (7.0% vs. 10.6%) to the drilling LAH group. There were no significant differences in pregnancy outcomes between subgroups defined based on age (older or younger than 35 years) or ZP thickness (greater or less than $17{\mu}m$) according to the LAH method. Conclusion: The present study demonstrated that partial ZP thinning or drilling resulted in similar outcomes in implantation and pregnancy rates using thawed embryos, irrespective of women's age or ZP thickness.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.9-20
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• 1999

The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.287-292
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• 1998

The objective of this study was to test whether ZP drilling using a 1.48$\mu$m diode laser beam on mouse IVF embryos becomes effective the hatching and normal in vivo development, as a preliminary test for obtaining the additional proof that the 1.48$\mu$m diode laser could be used safely for human applications. The results obtained in this experiment were as follows: when the hatched rates of mouse embryos by laser ZP drilling according to the embryonic stage were examined until 72 hr (in case of blast tocyst: day 4 after IVF) or 120 hr (in case of 4-cell: day 2 after IVF) after treatment, the d data of laser drilled blastocysts (81.8%) was significantly higher than those of control (hatching blastocyst: day 4 after IVF) (54.2%) and laser drilled 4-cell embryos (45.5%) (p<0.05). When the effect of laser drilling on implantation rates following embryo transfer in day 3 synchronized pseudopregnant recipients was examined, the l laser drilled group (48.7%) was slightly higher than that of control group (43.6%). In addition, when the several pregnant mice delivered in two groups were analysed their chromosomal normality and tested reproductive ability, all p pups were presented normal chromosomal number (n=40) and showed normal growth and reproductive ability. Therefore, these results dem-onstrated that ZP drilling using a 1.48$\mu$m diode l laser can increase the embryo hatching and ind duce the normal pregnancy of mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.1
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• pp.55-64
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• 2005

Objective: Human infertility clinics have been faced the demand for improving clinical results. The purpose of this study was to evaluate the effect of microsurgical removal of damaged blastomeres (DB) in frozen-thawed embryos on the clinical outcomes. Methods: From January 2003 to May 2004, out of 258 thawing ET cycles were divided into three groups: Group-1 (n=46): Intact cleavaged embryos after thawing. Remained cycles with embryos containing DB were randomly divided into two groups. Group-2 (n=102): Drilling zona pellucida (ZP) of frozen-thawed embryos by acidified Tyrode's solution. Group-3 (n=110): Drilling ZP and removal of DB. Embryos after microsurgical manipulation were transferred into the uterus of patients. Results: Clinical profiles and the mean number of transferred embryos among three groups were not different. Pregnancy and implantation rates were similar in three groups. It were 30.4% and 9.3% in Group-1, 29.4% and 7.8% in Group-2, and 26.4% and 7.6% in group-3, respectively. Miscarriage rate in Group-3 (37.9%) was slightly higher than those in Group-1 and Group-2 (14.3% and 23.3%), but it was not statistically significant. Conclusion: Intact cleaving embryos after DB removal showed higher potent of pregnancy and implantation. We could not find any improvement of clinical outcome by removal of DB in frozen-thawed embryos.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.213-221
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• 2001

This study was conducted to examine in vitro developmental ability of porcine embryos after somatic cell nuclear transfer. The porcine ear fell was cultured in vitro for confluency in serum-starvation condition(TCM-199 + 0.5% FBS) far 3~6 days of cell confluency. The zona pellucida of IVM oocytes were partially drilled using laser system. Single somatic cell was individually transferred into enucleated oocytes. And the reconstructed embryos were electrically fused(single DC 1.9kv/cm, 30$\mu$ sec) with 0.3M mannitol. After electrofusion, embryos were activated(single AC 5v/mm, 5sec) and cultured in HCSU-23 medium containing 10% FBS at 39$^{\circ}C$, 5% $CO_2$ in air for 6 to 8 days. The fusion rate of donor cells was 45.6, 36.8 and 46.1% in 3~4, 5~6 days of serum starvation and non serum starvation(N-S), and were 52.7. 53.0 and 51.7% in 1~2. 5~6 and 13~14 passages of donor cell culture, respectively. No significant difference was found in the fusion rate of donor cells by the duration of serum starvation treatment or the number of donor cell passages. By the size of donor cells, however, the fusion rate was significantly higher(P<0.05) for reconstructed embryos derived from 25r $\mu$m $\geq$ site of donor cells (65.3%) than that of 25~30$\mu$ m(42.5%) or 30$\mu$ m(45.5%)$\leq$ cells. The cleavage rate was significantly (P<0.05) higher in 3~4 darts of serum starvation treatment(67.1%) than that in N-S (50.7%) or 5~6 days of starvation(57.1%). The activation rate by the size of donor cells in fused oocytes was 56.5, 68.8 and 58.5%, respectively, and was not significant.