Embryonic Toxicity Test of Medical Devices for Human Assisted Reproductive Technology / 中国医疗器械杂志

OBJECTIVE@#To introduce the test methods of embryo toxicity applied to medical devices for human assisted reproductive technology (ARTMD), and provide the evaluation reference.@*METHODS@#The embryo toxicity test methods of ARTMD were summarized, and the key procedures and challenges in their safety evaluation were also discussed.@*RESULTS@#Establishing sensitive and stable test system is important to guarantee the safety and efficacy of ARTMD.@*CONCLUSIONS@#It remains development opportunities in improving sample preparation, extending test technology and expending evaluation method.

Advances in the study of the murine hematopoiesis during the early stage of embryogenesis / 解剖学报

The development of the murine embryonic hematopoietic system occurs in spatially and temporally distinct waves, which it is described as three waves so far-primitive hematopoiesis, bipotential erythroid-myeloid progenitors (EMPs) generation and long-lived transplantable hematopoietic stem cells (HSCs) maturation from their precursors and differentiation toward all the adult lineages. The latest point is that HSC-independent hematopoietic lineages are produced in the primitive wave and definitive progenitor wave in the early mammalian embryo, such as primitive erythrocytes or EMPs. The HSC-dependent phase of hematopoietic development produces all the adult lineages derived from HSCs. In this review, the recent studies on the development of hematopoietic cells and HSCs in the yolk sac and aorta-gonad-mesonephron region (AGM) region at cellular and molecular level will be summarized to provide an integrated model of developmental hematopoiesis, although multiple hematopoietic sites are involved in embryonic hematopoiesis. It may offer new insights into the characteristics and its underlying mechanism of hematopoiesis at the early stage of embryogenesis.

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.

Investigating the Potential of Nigella Sativa and Thymoquinone in Salvaging the Embryo from Effects of Toxic Paternal Exposure to Cyclophosphamide

Background: Exposure to cyclophosphamide (CPA) for cancer treatment results in over-production of reactive oxygen species and oxidative stress thus affecting the DNA in male germ cell inducing sperm defects. Our goal is to assess the potential effects of Nigella sativa extract (NSE) and thymoquinone (TQ) on sperm and embryo quality following fertlization of sperm produced from germ cells which have been exposed to the damaging alkylating effects of CPA. Methods: Thirty six male ICR mice were divided into six groups; (I) Vehicle-treated control (normal saline), (II) CPA-only, (III) TQ-only, (IV) NSE-only, (V) CPA followed by TQ and (VI) CPA followed by NSE. Treatment with 200mg/kg CPA and 10mg/kg of both NSE and TQ were given by intraperitoneal injection. Animals were sacrificed at 33 days by cervical dislocation and sperm from caudal epidydymis were taken for analysis and in vitro fertilization (IVF) with eggs from untreated female. Fertilization rates and embryo development were monitored for 5 days. The result were analysed by using SPSS 16.Results: TQ and NSE supplementation to CPA-exposed male mice have no significant effect (p>0.05) on the total number of sperm if compared to CPA-only exposed mice. NSE and TQ supplementation have been shown to have significant effect (p<0.05) on the percentage of motile sperm as well as the number of abnormal sperm. Four types of abnormalities of the sperm were found which includes folded sperm, amorphous, banana-like and the head lacking of the usual hook. Finally, the embryo quality shows a significant improvement by the supplementation of TQ and NSE to CPA-exposed male mice (p<0.05). Conclusion: Overall, both NSE and TQ have indicated chemopreventive potential against the cytotoxicity of cyclophosphamide on the reproductive capacity and fertility.

Analysis of factors affecting the standardization of in vitro mouse embryo test / 中国比较医学杂志

Infertility has become a global problem affecting human reproductive health.As an important treatment for infertility, assisted reproductive technology has made great progress over the past few decades.Rapid development has also taken place in medical devices for human assisted reproductive technology.It is imperative to establish the risk management and safety evaluation system of these products.In 2016, the industry standard YY/T 1434-2016 Human in vitro Assisted Reproductive Technology With Medical Equipment in vitro Mouse Embryo Test was officially released.In this paper, the key notes and elements of this in vitro mouse embryo test are briefly reviewed.

Construction of induced mouse pluripotent stem cells by piggyBac transposon and its identification / 吉林大学学报(医学版)

Objective:To investigate the effect of piggyBac transpon,as a carrier of four defined transcription factors Oct4,Sox2,Klf4 and c-Myc,in the reprogramming of mouse embryonic fibroblasts (MEFs)to induced pluripotent stem cells (iPSCs).Methods:The MEFs were isolated from Oct4-GFP fetal mice and transfected by piggyBac transposon with four factors (Oct4,Sox2,Klf4 and c-Myc).The morphological changes of clones were traced with microscope during the process of induction.The chromosomes were analyzed to evaluate the karyotypic variation of iPSCs.The mRNA expressions of Oct4, Nanog and FGF4 associated with embryonic stem cells (ESCs)in the iPSCs of mice were tested by RT-PCR;the protein expressions of SSEA-1,Nanog and Alkaline phosphatase in the iPSCs of mice were determined by flow cytometry,immunofluorescence and AP staining.The iPSCs were transplanted into the NOD-SCID mouse groin,4 weeks later,the teratomas were removed for HE staining and the differentiation of tissue was observed.Results:The iPSCs were successfully obtained from MEFs by piggyBac carrying Oct4,Sox2,Klf4,and c-Myc.The round or oval iPSCs clones were similar to ESCs with clear boundry and large dense nuleus.The iPSCs showed the normal karyotypic and expressed the marker genes (Oct4,Nanog and FGF4)and proteins (SSEA-1,Nanog and AP)of ESCs.Teratomas containing three germ layers were formed in NOD-SCID mice after tanspalantation of iPSCs.Conclusion:The iPSCs are reprogrammed from MEFs by piggyBac transposon with four transcription factors-Oct4,Sox2,Klf4 and c-Myc,and the iPSCs with normal karyotype possess the characteristics of ESCs.

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.

Effect of cell-penetrating peptide-conjugated estrogen-related receptor beta on the development of mouse embryos cultured in vitro / 대한생식의학회지

OBJECTIVE: Estrogen related receptor beta (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotency-related genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. METHODS: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without 2 microg/mL CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. RESULTS: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. CONCLUSION: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture

OBJECTIVE: This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. METHODS: A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P < 0.05). The rates of embryonic development after 48 hours (morula< or =) and 96 hours (blastocyst< or =) were significantly higher in 20% SSS and 10% SPS than in 20% hFF supplementation (P < 0.05). And the rates of embryonic development after 96 hours (hatching blastocyst< or =) were significantly higher in 10% SPS (94.5%) than in 20% SSS (82.6%) and 20% hFF supplementation (68.5%). The rates of embryonic development according to storage period of the SF-VCM supplemented with 10% SPS showed no significant difference between control, 2 weeks and 4 weeks group. However developmental rate in 6 weeks storage group was significantly lower than other groups. CONCLUSION: The rate of embryonic development after 96 hours (hatching blastocyst< or =) was significantly higher in SF-VCM supplemented with 10% SPS. And storage period of media up to 4 weeks did not affect on embryonic development.

Differential Expressions of Synaptogenic Markers between Primary Cultured Cortical and Hippocampal Neurons

Primary dissociated neuronal cultures are widely used research tools to investigate of pathological mechanisms and to treat various central and peripheral nervous system problems including trauma and degenerative neuronal diseases. We introduced a protocol that utilizes hippocampal and cortical neurons from embryonic day 17 or 18 mice. We applied appropriate markers (GAP-43 and synaptophysin) to investigate whether neurite outgrowth and synaptogenesis can be distinguished at a particular period of time. GAP-43 was found along the neural processes in a typical granular pattern, and its expression increased proportionally as neurites lengthened during the early in vitro period. Unlike GAP-43, granular immunoreactive patterns of synaptophysin along the neurites were clearly found from day 2 in vitro with relatively high immunoreactive levels. Expression of synaptic markers from cortical neurons reached peak level earlier than that of hippocampal neurons, although neurite outgrowths of hippocampal neurons were faster than those of cortical neurons. The amount of peak synaptic markers expressed was also higher in cortical neurons than that in hippocampal neurons. These results strongly suggest the usefulness of primary cultured neurons from mice embryos for synaptic function and plasticity studies, because of their clear and typical patterns of morphology that establish synapses. Results from this study also suggest the proper amount of time in vitro according to neuronal types (cortical or hippocampal) when utilized in experiments related with synaptogenesis or synaptic activities.

Effect of vitrification on viability and chromosome abnormalities in 8-cell mouse embryos at various storage durations

This study was desµgned to investµgate the effect of vitrification and post-thaw survival and chromosomal aberrations caused by vitrification of vitrified 8-cell mouse embryos in comparison with a controligroup. To this purpose the survival rate and the frequency of chromosomal aberrations were assessed in frozen-thawed 8-cell mouse embryos after various storage durations in the presence of ethyleneiglycol as cryoprotectant. eight-cell mouse embryos were obtained from NMRI mice 3 days after mating. Retrieved embryos were transferred to vitrification solution containing ethyleneiglycol as cryoprotectant, then transferred into a vitrification straw using standard technique, and vitrified in liquid nitrogen. Sixigroups of embryos according to storage duration (24 hours, 1 and 2 weeks, 1-6 months) were frozen. After appropriate storage periods embryos were thawed and studied for their viability 4-6 hours after thawing and intact embryos were transferred to fresh medium containing colcemid. After 48 hours, the embryos were fixed and studied for their chromosome abnormalities using Tarkowsky's drying technique. Results indicate that freezing affects the viability and chromosome structure of embryos when compared with the controligroup. Furthermore increasing the storage duration reduces the viability and increases the chromosome aberrations of embryos (such as aneuploidy and polyploidy). This result mµght indicate that the effects of vitrification on the cytoskeleton or other cellular organelle mµght produce chromosomal alterations leading to cell death.

The effectiveness of ethylene glycol as cryoprotectant in mouse embryo freezing with slow freezing method / 대한산부인과학회지

OBJECTIVE: We intended to know how the cryoprotectant ethylene glycol (EG) would affect the outcome of the embryo development when used in slow freezing method. And to know if there is any difference in the outcome of frozen-thawed embryos according to freezing methods and the timing. METHODS: We used 5-6 weeks old ICR female mice and T6 containing 0.4% BSA for basic culture media. The embryos at the developmental stages of 1-cell, 8-cell and blastocyst were cryopreserved respectively by slow freezing method using EG, propylene glycol (PROH), and glycerol as a cryoprotectant. We also compared the results of slow freezing and vitrification methods with the same cryoprotectant, EG. And finally, we evaluated the quality of blastocysts by counting the cell numbers in each group. RESULTS: The post-thaw embryo development were better in EG group when they were frozen at 1-cell and blastocyst stage (P<0.05). Although there were no differences in the recovery rate, the survival rate in vitrification group was significantly higher (P<0.05). Post-thaw embryo development to morula and blastocyst were better in vitrification group when frozen at 1-cell embryo (P<0.05), not at 8-cell and blastocyst group. The cell counts of blastocyst derived from 1-cell stage frozen EG group were significantly increased than that of PROH-glycerol groups (P<0.05), however, there was no difference between the two freezing methods. CONCLUSION: These results suggest that EG may be advantageous comparing with the conventional cryoprotectants, PROH and glycerol in slow freezing method for mouse embryo cryopreservation. In terms of freezing method, vitrification is better than slow freezing.

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.

Comparative study on the development of frozen-thawed mouse embryo using vitrification at each developmental stage / 대한산부인과학회잡지

OBJECTIVE: To find out the optimal freezing time in terms of developmental stage of mouse embryo and the effectiveness of vitrification method for freezing them. METHODS: Superovulation induction was performed using PMSG and hCG. Total 1,437 mouse embryos (vitrification group: 743, slow-freezing group: 694) were obtained and cultured with the T6 containing 0.4% BSA medium. Each developmental stage of embryos (1-, 2-, 4-, 8-cell, morula and blastocyst) were cryopreserved by vitrification and also by slow freezing method for comparison of the results. After thawing, the recovery rate, the survival rate and the blastocyst developmental rate were analysed and compared in two different settings. Student's t-test was used for statistical analysis. RESULTS: The survival and developmental rates at all subgroups of vitrification method are significantly higher than those of slow-freezing groups, but not in the recovery rates. In vitrification group, the survival rate and the blastocyst developmental rate are highest when frozen at morula stage, 98.4% and 86.4%, respectively. In slow-freezing group, the survival rate is also highest when frozen at morula stage, 87.2%, and the blastocyst developmental rate is highest when frozen at 8-cell stage, 78.1%. CONCLUSION: The vitrification method is more efficient for mouse embryo freezing compared with slow freezing one. Among various developmental stages of mouse embryos, morula stage seems to be the optimal stage for cryopreservation, whatever the freezing method applied. Therefore, we recommend embryo freezing at morula stage by vitrification method.

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.

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.

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.

Effect of Human Oviduct Epithelial Cells and Vero Cell on Early Mouse Embryonal Development In Vitro / 대한산부인과학회잡지

OBJECTIVE: The purpose of the study was to determine the effects of co-culture with oviductal epithelial cells and Vero cells on mouse embryo. METHOD: For the control group, mouse embryos were cultured alone in Ham's F-10 with 10% FBS. Subcultured oviductal epithelial cell and Vero cell were cocultured in Ham's F-10 with 10% FBS with the mouse embryo and used as the treatment group. Development of mouse embryos were observed. Result: The development rate and hatching rate of embryos that cocultured with oviductal epithelial cell and Vero cell was significantly higher (p<0.05) than control group. When subcultured oviductal epithelial cells were co-cultured with mouse embryo, there was no significant difference in development rate and hatching rate among subculture step. When oviductal epithelial cells that have been frozen-thawed were co-cultured with mouse embryo, there was no significant difference in development rate and hatching rate among subculture step. No statistical significance was seen in the development rate and hatching rate between subcultured oviductal epithelial cells and frozen-thawed oviductal epithelial cells when cocultured with mouse embryo, Vero cells and frozen-thawed when cocultured with mouse embryo, and Vero cells and oviductal epithelial cells when cocultured with mouse embryo. CONCLUSION: Oviductal epithelial cells and Vero cell may have a stimulatory role in early mouse embryonal development compared to control in vitro. As well, there is no significant difference in development rate and hatching rate among subculture step, when early mouse embryo was cocultured with cells that subcultured and frozen-thawed.

Study on Development of Mouse Preimplantation Embryos in Culture Media with Different Composition of Energy Sources / 대한산부인과학회잡지

OBJECTIVE: The objective of this study was to examine the effect on development of mouse preimplantation embryos in culture media with different composition of energy sources in vitro culture. METHODS: Two hundred and seventy one two-cell embryos were cultured in four different culture system for 96 hours. Group I (n=61) was cultured in DMEM-G (DMEM with glutamine) only, groupII (n=64) was cultured in DMEM-GGP (DMEM with glutamine, glucose and pyruvate) only, group III (n=72) was cultured for 48 hours in DMEM-G and then transferred to DMEM-GGP and group IV (n=74) was cultured for 48 hours in DMEM-GGP and then transferred to DMEM-G. Development of embryos in each group was observed every 24 hours. RESULTS: After 24 hours, the rate of development > or = 3-cell was significantly higher in groupII (87.5%) and IV (86.5%) compared with group I (59.0%) and III (62.5%). After 48 hours, the rate of development into > or = morula stage was significantly higher in GroupII (79.7%) and IV (86.5%) compared with group I (34.4%) and III (37.5%). After 72 hours, the rate of development into blastocyst was significantly higher in group IV (74.3%) compared with group I (49.2%) and III (45.8%). After 96 hours, the rate of development into > or = expanded blastocyst was significantly higher in group IV (70.3%) compared with group I (32.8%),II (53.1%), and group III (40.3%). CONCLUSION: Mouse preimplantation embryos development was the most effective in culture system with DMEM-GGP for 48 hours and then transferred to DMEM-G.

Effect of Partial Laser Assisted Hatching on Mouse Embryos / 대한불임학회지

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 micrometer 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 20~22 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+/-6.4 micrometer) was smaller than that of control group (122.2+/-5.0 micrometer), but larger than that of c-LAH group (102.2+/-2.7 micrometer). Zona pellucida thickness at hatching time of p-LAH group (6.4+/-0.9 micrometer) was thicker than that of control group (4.5+/-1.5 micrometer), but thinner than that of c-LAH group (10.0+/-0.8 micrometer). 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.