• Journal of Genetic Medicine
• /
• v.7 no.2
• /
• pp.125-132
• /
• 2010

Purpose: Preimplantation genetic diagnosis (PGD), also known as embryo screening, is a pre-pregnancy technique used to identify genetic defects in embryos created through in vitro fertilization. PGD is considered a means of prenatal diagnosis of genetic abnormalities. PGD is used when one or both genetic parents has a known genetic abnormality; testing is performed on an embryo to determine if it also carries the genetic abnormality. The main advantage of PGD is the avoidance of selective pregnancy termination as it imparts a high likelihood that the baby will be free of the disease under consideration. The application of PGD to genetic practices, reproductive medicine, and genetic counseling is becoming the key component of fertility practice because of the need to develop a custom PGD design for each couple. Materials and Methods: In this study, a survey on the contents of genetic counseling in PGD was carried out via direct contact or e-mail with the patients and specialists who had experienced PGD during the three months from February to April 2010. Results: A total of 91 persons including 60 patients, 49 of whom had a chromosomal disorder and 11 of whom had a single gene disorder, and 31 PGD specialists responded to the survey. Analysis of the survey results revealed that all respondents were well aware of the importance of genetic counseling in all steps of PGD including planning, operation, and follow-up. The patient group responded that the possibility of unexpected results (51.7%), genetic risk assessment and recurrence risk (46.7%), the reproduction options (46.7%), the procedure and limitation of PGD (43.3%) and the information of PGD technology (35.0%) should be included as a genetic counseling information. In detail, 51.7% of patients wanted to be counseled for the possibility of unexpected results and the recurrence risk, while 46.7% wanted to know their reproduction options (46.7%). Approximately 96.7% of specialists replied that a non-M.D. genetic counselor is necessary for effective and systematic genetic counseling in PGD because it is difficult for physicians to offer satisfying information to patients due to lack of counseling time and specific knowledge of the disorders. Conclusions: The information from the survey provides important insight into the overall present situation of genetic counseling for PGD in Korea. The survey results demonstrated that there is a general awareness that genetic counseling is essential for PGD, suggesting that appropriate genetic counseling may play a important role in the success of PGD. The establishment of genetic counseling guidelines for PGD may contribute to better planning and management strategies for PGD.

• Clinical and Experimental Reproductive Medicine
• /
• v.33 no.1
• /
• pp.25-33
• /
• 2006

Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

• Clinical and Experimental Reproductive Medicine
• /
• v.23 no.1
• /
• pp.25-32
• /
• 1996

This work has been carried out to examine the number of Total, ICM and TE cells of F1 mouse blastcysts at day 4 after IVF by differential labelling of the nuclei with polynucleotide-specific fluorochromes and to obtain a fundamental information of preimplantation mouse embryo development. Blastocysts produced by superovulated B6CBA F1(C57BL/${\times}$CBA) eggs were inseminated with $1{\times}10^6$spermatozoa/ml and cultured in M16 medium at $37^{\circ}C$, 5% $CO_2$ incubator for 95hrs. Blastocysts were classified as early, middle, expanded and hatching stage according to the developmental morphology; blastocoel expansion and zona thickness. The results obtained in these experiments were summarized as follows; 1) The development rate of blastocysts at 95hrs after IVF was 86.7% and classified blastocysts to early, middle, expanded and hatching were 16.3%, 18.9%, 10.5% and 40.9%, respectively. 2) The numbers of total blastomere using bisbenzimide in the classified blastocysts to early, middle, expanded and hatching were 35.6${\pm}$1O.4, 49.4${\pm}$8.6, 60.8${\pm}$1O.7 and 62.7${\pm}$13.9, respectively. 3) In ICM and TE cell number by using differential labelling with polynucleotide-specific fluorochrome in the classified blastocysts to early, middle, expanded and hatching; ICM numbers were 9.6${\pm}$3.0, 13.6${\pm}$3.9, 16.0${\pm}$3.3 and 19.5${\pm}$4.6, respectively and TE cell numbers were 30.6${\pm}$5.1, 39.9${\pm}$5.8, 42.2${\pm}$8.1 and 43.7${\pm}$11.1, respectively. These results showed the same increase pattern according to development advance level. Also, when compared with the results of total count were obtained between bisbenzimide only and differential labelling, both of them showed the same increase pattern according to development level and at the same time their cell numbers were almost the same. So, rapid and simple cell count method using differential labelling can be used for the examination of later preimplantation development or as an indicator of embryo quality according to the variables of culture conditions.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.56-56
• /
• 2003

Beta-catenin is very important in early development including involvement in cell adhesion, cell signaling, and developmental fate specification. Cell-cell interaction is an important process during mammalian embryonic development. In preimplantation embryos, embryonic compaction is the process of increased cellular flattening and adhesion of junctional complexes and results in a polarized distribution. Beta-catenin is associated with embryonic compaction in mammals. Here, we examined the relationship between beta-catenin expression and compaction in porcine embryos derived from in vitro fertilization. First of all, we investigated beta-catenin expression in each embryonic developmental stage and also focused on expression pattern according to full, partial and non-compaction at morula stage. We used the immunocyto-chemical method in this research. To confirm compaction affects on the embryonic development, we compared between compaction and developmental rates to the blastocyst. The result showed that compaction and non-compaction rates were 14.6% and 63.8% at 4 days after IVF, respectively The developmental rates to the blastocyst and their total cell number were 50.9% vs 36.4% and 41.4$\pm$11.5 vs 26.8$\pm$12.7 in compaction and non-compaction groups. Although no difference was detected in the ratio of ICM to total cells between two groups, total cell number of the blastocysts in compaction group was superior to that of the blastocysts in non-compaction group (P<0.05). Expression of beta-catenin appeared in the boundary of membrane surface between blastomeres in 2- and 4-cell stage, and observed irregular pattern from 8-cell to blastocyst stage. We also investigated beta-catenin expression pattern according to the degree of compaction in the 3 groups; full, partial (>50%) and non-compaction. The expression signal in fully compacted embryos was stronger than those of partial and non-compacted embryos. Especially, beta-catenin expression appeared various patterns in morula stage suggesting the aberrant distribution of beta-catenin is affected by compaction patterns. Our results suggest that abnormal beta-catenin expression was affected by embryo quality and further development in porcine embryos in vitro.

• Reproductive and Developmental Biology
• /
• v.38 no.3
• /
• pp.115-121
• /
• 2014

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was evaluated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental competence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, $0.1{\mu}g/mL$, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of penetrated oocytes but ROS intensity was significantly decreased by $0.01{\mu}g/mL$ compared with other groups (p< 0.05). Moreover, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supplementation of Humulus japonicus extract in IVF medium may have important implications for improving early embryonic development in bovine embryos.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.5
• /
• pp.711-717
• /
• 2007

In this study, two cell types from porcine females, namely fetal fibroblasts (pFFs) and adult ear fibroblasts (pAEFs) and two passages (3-4 and 7-8) were investigated by evaluating the development rate, blastocyst cell number and the incidence of apoptosis. No significant differences were observed in the cleavage rates of cloned and IVF embryos. The blastocyst rates between the embryos cloned with pFFs ($15.1{\pm}3.2$) and pAEFs ($10.4{\pm}2.6$) did not differ significantly but was significantly (p<0.05) lower in pAEFs than that in IVF ($22.5{\pm}4.5$) embryos. Total cell number in pFFs ($28.4{\pm}4.3$) and pAEFs cloned blastocysts ($24.2{\pm}5.1$) was significantly (p<0.05) lesser than IVF control ($35.4{\pm}3.2$). Apoptosis rates between cloned blastocysts differed significantly (p<0.05) and were significantly (p<0.05) higher than IVF embryos. The blastocyst rates between the cloned embryos cloned with different cell passages did not differ significantly but in embryos cloned with 7-8 cell passage was significantly (p<0.05) lower than the IVF control. Apoptosis signals were detected in IVF and cloned embryos as early as day 3 and the rates of apoptosis increased concurrently with the embryo development. In conclusion, high apoptosis during in vitro preimplantation development resulted in low development rate and total cell number of cloned embryos. Moreover, based on the apoptotic incidence in cloned blastocysts, fetal fibroblasts are more suitable for production of cloned embryos in porcine.

• Development and Reproduction
• /
• v.5 no.1
• /
• pp.17-21
• /
• 2001

This study was conducted to investigate the expression pattern of Trypsin-like enzyme and the effect of a trypsin inhibitor(benzimidine) on hatching process during in-vitro culture of mouse preimplantation embryos. The Trypsin-like enzyme was identified by rhodamine-conjugated Trypsin substrate probe. The expression of trypsin-like enzyme was firstly detected at the late morula stage, and the enzyme was uniformly localized in the trophectoderm of late blastocysts. Especially, intense fluorescence was observed in the blebbing area of hatching blastocysts. Bisbenzamidine, contained in culture media, did not alter embryonic development from 4-cell stage to the expanded blastocyst but decrease the hatching rate in ImM concentration (15.8% vs 89.7%, p<0.02). In the treatment of bisbenzimidine (5mM) for 12 hours according to the embryonic stage of mouse, the hatching rate of control (83.0%) and treatment in late blastocysts (8.7%) were significantly (p<0.01) different. From these results, we suggested that the hatching enzyme having trypsin-like activity was localized from the late morula stage, and the hatching process by this enzyme was activated in the late blastocyst stage of mouse embryos.

• Journal of Embryo Transfer
• /
• v.19 no.2
• /
• pp.133-145
• /
• 2004

Serum-contain is commoly used for the production of in vitro-derived bovine embryos. However, were biological activity of serum varies from lot to lot, time consuming to choose better serum with good quality and risks of virus, bacteria and mycoplasma infection. This study established serum-free culture systems of in vitro embryo development to efficiently obtain a large number of blastocysts from ovaries of Hanwoo and oocytes maturation, cell number, tlerance of cryopreservation. Secondly, serum-contain medium is suspected of contributing to the large calf size, dystocia, cersarean sections, calf mortality and confirmed these blastocysts are high quality in terms of cyotolerance, high rates of pregancy and normal birth. For these reasons, Culture media (IVMD101 and IVD101) designed specifically for the preimplantation bovine embryo are rather simplistic, being based on salt solutions with additional energy substrates and growth factors. An improved serum-free medium (IVMD101) was developed for bovine oocytes maturation in vitro. Proportions of embryos developing to the blastocyst stage cultured in both IVD101(32.4%) and IVD101(34.5%) serum-free media were higher than in TCM199+10% FBS(12.4%) serumcontaining medium. Futhermore, the cell numbers per blastosyst obtained in the serum-free media were superior to those of blastocysts developed in serum-supplemented medium. Also, cell numbers of blastocysts obtained in the serum-free media were similar with blastocysts derived in vivo. Survival rate blastocysts after 24 hr incubation after thawing, the blastocysts cultured in both IVD101(94.5%) and IVD101(95.8%) serum-free media were higher than in TCM199+10% FBS (52.5%) serum-containing medium. After 72 hr incubation after thawing, hatching rates of blastocysts developed in IVD101(78.4%) and IVMD101(83.7%) were sighnificantly higher than that developed in the serum-supplemented medium(32.0%). The pregnancy rates almost not different between fresh blastocysts(38.2%) and frozen blastocysts(34.9%). The results suggested that the improved serum-free media(IVMD101 and IVD101) offer several advantages over culture in serum-cotaining medium, including increased rates of blastocyst formation and high cel numbers. Additionally, the survival and hatching rates of embryos product in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containg medium and useful for the production of high quality bovine embryos for cryo-preservation. These improved serum-free media are beneficial not only for the study of the mechanisms of early embryogenesis but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

• Korean Journal of Veterinary Research
• /
• v.29 no.2
• /
• pp.123-128
• /
• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• Reproductive and Developmental Biology
• /
• v.31 no.1
• /
• pp.49-53
• /
• 2007

Astaxanthin is a kind of carotenoid compounds, having a antioxidant and anti-inflammatory activities. The antioxidative mechanism by which carotenoid scavenge free radicals has been clearly elucidated, but has not tried for the development of mammalian preimplantation embryo. This study was conducted to investigate the antioxidative effect of astaxanthin on in vitro development of porcine in vitro fertilized embryos. Porcine embryos derrived from in vitro fertilization (IVF) were cultured in 5% $CO_{2} in air at $38.5^{\circ}C$ in PZM-3 medium supplemented with different dosages of astaxanthin ($0,\;1,\;5\;and\;10{\mu}M$) and taurine (0, 1, 2.5 and 5 mM) as a positive control, and execute to compare the effects of various antioxidants such as taurine, melatonin and asculatin on in vitro development. The proportions of embryos developed to the blastocyst stage were increased when $1\;and\;5\;{\mu}M$ of astaxanthin (26.6 and 23.4%, respectively) and 1 and 2.5 mM taurine (25.8 and 26.4%, Respectively) were supplemented, compared to controls (p<0.05). Also, various antioxidant-treated groups were significantly higher rates of blastocysts (astaxanthin, 27.4%; taurine, 29.1%; melatonin, 26.8%; aesculetin, 27.9%, respectively) than control (18.8%). There was no difference in mean cell number of blastocysts between antioxidants and control. This result indicates that astaxanthin has an antioxidant feature when porcine IVF embryos were cultured in vitro.