The Effect of Vero Cell Coculture on the Development of Mouse Embryos Exposed to Monoclonal Antibodies Specific for Mammalian Heat Shock Protein 60

Heat shock proteins (HSP) have been identified as an important factor of a very complex and highly conserved cellular defense mechanism to preserve cell survival under adverse environmental conditions. HSP 60 are immunodominant antigens of microbe such as Chlamydia trachomatis and have a potentiality to become a target antigen due to antigenic similarity between chlamydial and human HSP. This study was conducted to investigate the effects of Vero cell coculture to anti-HSP 60 on the early mouse embryo development in vitro. The 2-cell mouse embryos (ICR) were cultured and mouse embryo development was observed every 24 hr for 3 days. 45% and 22.1% of the embryos cultured in Ham's F-10 plus anti HSP 60 with Vero cells developed to the 4- to 8- cell stage (day 1) and morular stage (day 2) as compared with 29.2% and 2.7% of those cultured without Vero cells respectively. But at day 3, the beneficial effect of Vero cells was not noted. These findings suggest that Vero cells have some roles to overcome the detrimental effect of anti-HSP 60 to some degree. These results suggest that Vero cells coculture will promote reproductive outcome in patient previously sensitized to microbial (e.g. Chlamydia trachomatis) HSP 60.

The Effects of Vero Cell Co-culture on the Expression of Bax and Bcl-2 Genes in the Mouse Embryo Development / 대한산부인과학회지

OBJECTIVE: To investigate the effects of Vero cell co-culture on the development of mouse embryo in vitro and the expression of bax and bcl-2 genes in the mouse embryo. METHODS: The 2-cell mouse embryos were obtained from oviduct of 5-6 weeks old mated female ICR mice superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The 2-cell embryos in Ham's F-10 medium supplemented with 10% FBS with or without Vero cells, were incubated at 37 degrees C in a 5% CO2 humidified air chamber respectively and we observed mouse embryo development and collected each group of embryos for the purpose of extraction of total RNA every 24 hours for 3 days. We carried out the RT-PCR to assess mRNA levels for bax and bcl-2 gene. RESULTS: The rate of embryo development of with Vero cell co-cultured group was 78.3%, 50.7%, 27.2% and that of without Vero cell co- cultured group was 60.5%, 29.3%, 20.7% respectively. Bax mRNA expression level of without Vero cell co-cultured group was significantly higher than that of with Vero cell co-cultured group at 24 hours. Bcl-2 mRNA expression level of Vero cell co-cultured group was significantly higher than that of without Vero cell co-cultured group at 72 hours. CONCLUSION: These findings suggested that Vero cell co-culture is beneficial in the development of mouse embryos and stimulates bax and bcl-2 gene expression.

Studies on the Effects of Monoclonal Antibodies to Mammalian Heat Shock Protein 60 (HSP 60) on Mouse Embryo Development In Vitro / 대한산부인과학회잡지

OBJECTIVE: To investigate the effects of antibodies to HSP 60 on the early mouse embryo development in vitro. METHODS: The 175 late 2-cell mouse embryos were obtained from 6-7 week old female ICR mice. 5-10 embryos were placed in each well. The embryos were incubated in the Ham's F-10 medium supplemented with 100 microgram/mL of monoclonal antibody to HSP 60 (66), monoclonal mouse IgG1 (55), and medium alone (54), respectively, at 37degrees C in a 5% CO2 humidified air chamber, and mouse embryo developments were observed daily. RESULTS: On day4, growth arrests were more prominent in anti-HSP 60 containing group compared to IgG1 containing group, medium only group (0% vs 16%, 14%), and these results were statistically significant (p=0.0032). Especially those inhibitory effects were observed in early stage of embryo development (day1) and these results were also statistically significant (31% vs 83%, 77%, p<0.0001). Moreover, we found out that cellular degenerations were more common in anti-HSP 60 containing group and this features were prominent on day2. CONCLUSION: Anti-HSP 60 elicited a strong growth inhibitory and degenerative effect on early mouse embryo development. These findings suggest that HSP 60 may exert a protective effect against mouse embryo degeneration or apoptosis.

The Effect of Equilibration Temperature and Time Prior to Cryopreservation on the Outcome of Cryopreservation of 2-Cell Mouse Embryos / 대한산부인과학회잡지

OBJECTIVE: One-cell human zygotes have been successfully frozen and thawed using 1,2-propanediol (PROH) during freezing and thawing. This study was performed to assess the effect of equilibration temperature and time on cryopreservation of 2-cell mouse embryos by investigating the equilibration temperature and time during initial PROH exposure prior to cryopreservation. MATERIALS AND METHODS: The late 2-cell mouse embryos were obtained from 5-6 week old ICR mice and were exposed to 1.5 M PROH in phosphate-buffered saline (PROH-PBS) at 37degrees C, room temperature (22- 24degrees C), and 4degrees C for 30 min. The PROH was washed off the 2-cell mouse embyos by incubating them for 5 min each in 1, 0.5, and then 0 M PROH-PBS in the order named. The 2-cell mouse embryos were subsequently cultured in Ham's F-10 medium and embryo development was assessed at 24, 48, and 96 hours. RESULTS: Incubation of 2-cell mouse embryos at 37degrees C for 30 min significantly impaired embryo development to blastocysts. Embryo development after exposure to PROH at 37degrees C for 10 min was 8.3% (P=0.047) and embryo development for 30 min was 5.4% (P=0.038). Incubation of 2-cell mouse embryos at room temperature or 4degrees C for up to 30 min did not significantly reduce embryo development. Cryosurvival of 2-cell mouse embryos exposed to PROH at room temperature or 4degrees C was similar. CONCLUSION: These findings suggested that pronanediol is toxic to 2-cell mouse embryos in a temperature- and time-dependent fashion. Cryopreservation of 2-cell mouse embryos after exposure at 4degrees C appears to be no better than after exposure at room temperature.

Studies on Pregnanacy Rate of Controlled Ovarian Hyperstimulation before Intrauterine Insemination in the Treatment of Unexplained Infertility / 대한산부인과학회잡지

OBJECTIVE: To determine which regimen for controlled ovarian hyperstimulation is the most effective in achieving pregnancy after intrauterine insemination in the treatment of unexplained infertility. MATERIALS AND METHODS: From March 1996 to February 2000, a total of 67 cycles of intrauterine insemination after controlled ovarian hyperstimulation were treated in 39 patients under 40 years old who diagnosed as unexplained infertility. Two methods of controlled ovarian hyperstimulation were used. The one is clomiphene citrate/hMG and the other is hMG only. These were compared the pregnancy rate respectively. RESULTS: Mean age of study group was 32+/-2.7 years old (28-38 years old) and mean duration of infertility was 46+/-17.8 months (15-96 months). The overall clinical pregnancy rate was 17.9% (12/67 cycle) per cycle and 30.7% (12/39 patient) per patient. According to the methods of controlled ovarian hyperstimulation, pregnancy rate was 16.7% (8/48 cycle) after clomiphene citrate/hMG used, 21.1% (4/19 cycle) after hMG only used. 4 cases of ovarian hyperstimulation syndrome developed (clomiphene citrate/hMG 1 case, hMG only 3 cases) and all of them were self-regressed. CONCLUSION: Compared with using hMG only as controlled ovarian hyperstimulation before intrauterine insemination, using clomiphene citrate/hMG was more effective regimen and considered as the first choice in the treatment of unexplained infertility.

Effects of Coculture of Vero Cells on the Development of Frozen-thawed Two-cell Stage ICR Mouse Embryos / 대한산부인과학회잡지

OBJECTIVE: The rate of developmental progression of frozen-thawed embryos is lower than that of nonfrozen embryos in mice, cows, humans and other mammalians. This study was designed and performed to evaluate the beneficial effects of coculture of Vero cells on the development of frozen-thawed two-cell stage embryos of ICR strain mice. MATERIASL AND METHODS: The late two-cell stage mouse embryos were obtained from oviducts of 5~6 week old mated ICR mice superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-cell stage mouse embryos were frozen slowly with 1,2-propanediol and sucrose as cryoprotectants and thawed rapidly, followed by stepwise dilution. The frozen-thawed embryos were cultured in Ham's F-10+10% Fetal Bovine Serum (FBS) basal culture medium with and without Vero cells. The rates of development in both groups were compared every 24 hours for 5 days. RESULTS: Vero cells did not significantly stimulate the rate of embryonal development compared to controls at 24 hours after culture, 124 (69.3%) and 68 (61.3%), respectively (p=0.161). On day 4, however, 55 (30.7%) cocultured embryos had developed to expanded-hatching blastocysts, which was the significantly higher number than that of the embryos in controls: 16 (14.4%) (p=0.002). In addition, more embryos in coculture developed to hatching-hatched blastocysts (43[24.0%]) compared to the controls (10[9.0%]) (p=0.001). CONCLUSION: Coculture of cryopreserved embryos after thawing with Vero cells seems to be an useful tool to remove the postthaw deleterious effects of freezing and to obtain better quality embryos appropriate for transfer. These beneficial effects of Vero cell coculture appear to become more prominent as the embryonic development progresses over time.

Vero Cell Co-culture Counteracts the Detrimental Effects of Hydrosalpinx Fluid on the Development of Mouse Embryos in vitro

Recent studies have suggested that the hydrosalpinx has a negative effect on pregnancy outcome, with markedly diminished implantation and increased early pregnancy loss. Fluid from the hydrosalpinx may leak into and accumulate in the uterine cavity. It is not clear, however if this creates a hostile local environment in the uterus for embryo implantation or exerts a direct embryotoxic effect. This study was conducted to investigate the detrimental effects of hydrosalpinx fluid (HSF) on the development of mouse embryos in vitro and to demonstrate whether Vero cells overcome these adverse effects. HSF was collected from three women with bilateral hydrosalpinx at the time of laparoscopic surgery. Collected fluid was centrifuged and the supernatant was frozen at -20degrees C. For co-culture, Vero cells were commercially obtained in a frozen state and cultured using Ham's F10 medium. Single-cell mouse embryos (B6CBAF1) were cultured for 5 days in 0, 0.4, 0.8, and 1.2% of HSF in media with and without Vero cells and examined daily to record the number of embryos reaching expanded blastocyst and hatching stage. Co-culture of mouse embryos with Vero cells at 0.8% HSF concentration significantly enhanced embryo development, but not at 1.2% hydrosalpinx fluid concentration. These results suggest that HSF is highly embryotoxic and Vero cells are likely to overcome these detrimental effects to some degree.

The Effect of Propofol (2,6-diisopropylphenol) and Thiopental Sodium on Fertilization and Early Embryo Development in Mouse IVF Model / 대한마취과학회지

BACKGROUND: Propofol and thiopental sodium are short acting drugs and used as intravenous anesthetics for oocyte retrieval. Anesthetics administered during oocyte retrieval can pass into the follicular fluids and exert a detrimental effect on oocyte fertilizability. The aim of this study was to investigate the exposed concentration and time effect of these drugs on fertilization and early embryo development in a mouse in vitro fertilization (IVF) model. METHODS: Mouse oocytes were exposed in vitro to propofol at 0 (control), 0.09, 0.45, 2.3, 4.5microgram/ml and thiopental sodium at 0 (control), 0.2, 1, 5, 10microgram/ml for 10, 30, 60 minutes, washed, and inseminated. Thereafter, fertilization was assessed. Subsequent in vitro development to the hatched embryo was monitored daily. RESULTS: We found a concentration and time dependent toxic effect of propofol on the fertilizability of oocytes and early embryo development. The fertilization rate of mouse oocytes exposed for 30 minutes to medium containing 0.09microgram/ml of propofol was significantly lower than the control. The fertilization and hatching rate of mouse oocytes exposed for 10 minutes to medium containing 0.45microgram/ml of propofol was not lower than the control. We did not find a toxic effect of thiopental sodium on fertilization, but the hatching rate of fertilized oocytes exposed to medium containing thiopental sodium was significantly lower than the control. CONCLUSIONS: We suggest that the oocyte retrieval procedure should be done as quickly as possible in order to limit the toxic effect of these anesthetics.