Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106), G2 (2*106) and G3 (4*106) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chi-square (P<0.05) and correlated with CASA and fluorescence data using Person Correlation (P<0.05). The IVF efficiency, cleavage and total blastocyst rates did not show any significant difference (P>0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P<0.05) without difference (P>0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P<0.05), without significant difference (P>0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106), G2 (2*106) e G3 (4*106) espermatozoides/mL. Às 18h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o Computer-Assisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P<0,05). A eficiência da FIV, taxas de clivagem e blastocisto total não mostraram diferença significativa (P>0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P<0,05), sem diferença (P>0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P<0,05), sem apresentar diferença significativa (P>0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0,05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106 ), G2 (2*106 ) e G3 (4*106 ) espermatozoides/mL. Às 18 h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o ComputerAssisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Effect of roscovitine on developmental competence of small follicle-derived buffalo oocytes

Background & objectives: The lower recovery of competent oocytes in buffalo species limits the commercialization of in vitro embryo production technology in field condition. In this context, pre-maturation of small follicle (SF)-derived oocytes with meiotic inhibition may be a promising alternative to obtain more number of competent oocytes. Thus, the present study was conducted with an objective to enhance the developmental potential of less competent SF-derived buffalo oocytes. Methods: All the visible follicles (used for aspiration) from buffalo ovaries were divided into two categories: large follicle (LF) (follicles having diameter ?6 mm) and SF (follicles of diameter <6 mm). The competence of LF and SF oocytes was observed in terms of brilliant cresyl blue (BCB) staining, cleavage rate, blastocyst rate and relative gene expression of oocyte and blastocyst competence markers. Thereafter, less competent SF oocytes were treated with 0, 12.5, 25, 50 and 100 mM doses of roscovitine (cyclin-dependent kinase inhibitor) to enhance their developmental potential. Results: Based on parameters studied, LF oocytes were found to be more competent than SF oocytes. Pre-maturation incubation of SF oocytes with roscovitine reversibly arrested oocyte maturation for 24 h to ensure the proper maturation of less competent oocytes. A significantly higher number of BCB-positive oocytes were noted in roscovitine-treated group than SF group. Cleavage and blastocyst rates were also higher in roscovitine-treated group. The relative messenger RNA expression of oocyte (GDF9, BMP15, GREM1, EGFR, PTGS2 and HAS2) as well as blastocyst (INF-?, GLUT1 and POU5F1) competence markers was significantly greater in roscovitine-treated group relative to SF group. Again, on comparison with LF group, these parameters depicted a lower value in the treatment group. Interpretation & conclusions: The findings of this study has revealed that pre-maturation incubation of SF-derived oocytes with 25 ?M roscovitine can improve its developmental competence and thus can be utilized to get maximum number of competent oocytes for better commercialization of in vitro embryo production technology in buffalo.

Estudio genético preimplantación para aneuploidias: resultados de la transición entre diferentes tecnologías / Preimplantation genetic testing for aneuploidies PGT-A: Results of the transition between different technologies

Resumen OBJETIVO Comunicar los resultados obtenidos del análisis del estudio genético preimplantación para aneuploidias en dos centros de reproducción asistida de México en un periodo de tres años, utilizando dos diferentes técnicas moleculares. MATERIALES Y MÉTODOS Estudio observacional, retrospectivo, en donde se reporta el resultado de blastocistos sometidos a preimplantación para aneuploidias durante 2014-2017, en dos centros de reproducción asistida (Ciudad de México y Guadalajara). RESULTADOS Se analizaron 404 blastocistos de 129 pacientes (edad promedio 39 ± 4 años). Los embriones se dividieron en dos grupos según la técnica aplicada: 76 por a-CGH y 328 por secuenciación de nueva generación. El porcentaje de embriones euploides fue de 33%. Las aneuploidias numéricas fueron las más frecuentes. Hasta la terminación del estudio se habían transferido 69 embriones euploides con tasas de implantación de 78% para secuenciación de nueva generación y de 57% para a-CGH. CONCLUSIONES La tasa de implantación reportada en este estudio fue mayor con el análisis de preimplantación para aneuploidias por secuenciación de nueva generación. Los resultados reportados en nuestra experiencia soportan la necesidad de favorecer una opción de transferencia de embrión único. Es importante reconocer los retos de las nuevas tecnologías y la necesidad de técnicas moleculares más sensibles.

Abstract OBJECTIVE Communicate the results obtained from the analysis of the preimplantation genetic study for aneuploidy in two centers of assisted reproduction in Mexico in a period of three years, using two differentmolecular techniques. MATERIALS AND METHODS Descriptive, retrospective study, which reports the blastocysts PGT-A results, over 2014-2017, in two Fertility Centers (Ciudad de México and Guadalajara). The embryos where divided in two groups by their molecular techniques studied: 76 by a-CGH and 328 by NGS RESULTS We analyzed a total of 404 blastocysts from 129 patients (mean age 39 ± 4 years), with two different molecular techniques: a-CGH and NGS. The euploid embryos percentage was 33%. The numerical aneuploidies were the most frequent. Up to the ending of the study, 69% of the euploid embryos had been transferred. The implantation rates were 78% for those analyzed by NGS and 57% with a-CGH. CONCLUSIONS The implantation rate was bigger with the PGT-A by NGS. Our results reported, supports a single embryo transfer policy. It is important to recognize the challenges of new technologies and the need for more sensitive molecular techniques.

Study on the spatial expression of trophectoderm cells in human embryonic prenatal blastocysts / 北京大学学报(医学版)

Objective:To study the spatial expression of trophectoderm cells in human embryonic preantral blastocysts.Methods:The study used Gardner score 5AA blastocysts harvested on day 6 after fertilization from assisted reproductive technology.Microcapsules were used to separate trophectoderm cells from the epidermal cells.Single-cell sequencing was performed.P ＜ 0.05 was calculated by unpaired t test,and the difference was 2 times.Here we determined,for the first time,global gene expression patterns in the polar/mural trophectoderm isolated from human blastocysts.Unsupervised hierarchical clustering analysis and gene ontology (GO) functional classification were performed using bioinformatics software.Differentially expressed genes were annotated by the Database for Annotation,Visualization and Integrated Discovery.Functions of differentially expressed genes were further annotated using encyclopedia of genes and genomes.Results:The results showed that there were up to 306 genes in the trophoblast cells and up to 75 genes in the trophoblast cells.Unsupervised cluster analysis of polar trophoblast cells and mural trophoblast cells were divided into two groups,belonging to different types and biological functions.Differences in gene function indicated that the biological functions of GO gene uptake genes were mainly transcription,energy metabolism,protein synthesis,transport,oxidative stress,ion transport,protein synthesis and transport,cell cycle regulation,actin growth,etc.They were mainly involved in ubiquitin-mediated protein hydrolysis,oxidative phosphorylation,Wnt signaling pathway,estrogen androgen metabolism and other signal pathways;wall trophoblast cells up-regulated gene GO biological function,which was mainly proteolytic metabolism,cell cycle arrest,apoptosis,activation of MAPK,carbohydrate transport,synaptic regulation,cell growth,calcium channel activation,positive B cell differentiation,T cell apoptosis and other biological functions,which were mainly involved in B cell receptor,T cell receptor,white blood cells cross-endothelial transplantation,VEGF expression,gap connection,GnRH secretion,apoptosis and other signaling pathways.Conclusion:The gene expression of blastocysts trophectoderm is revealed from the spatial dimension,indicating that differentiation of polar and mural trophectoderm of blastocysts is accompanied by differences between the two cell lineages,and the polar and mural trophectoderms are coordinated with each other and the blastocyst hatching and embryo implantation processes are finely adjusted.Further data analysis is expected to find the endogenous molecular specificity of the regulation of embryo implantation.

Doble transferencia embrionaria consecutiva D3/D5-6: una alternativa para mejorar las tasas de embarazo en pacientes sometidas a fertilización in vitro/transferencia embrionaria (FIV/TE)

Objetivo: Evaluar la doble transferencia embrionaria consecutiva D3/D5-6 (DTEC D3/D5-6) como alternativa para mejorar las tasas de embarazo en pacientes sometidas a un programa de fertilización in vitro/transferencia embrionaria (FIV/TE) que obtengan más de dos embriones de día 3 (D3), independientemente del número de intentos previos, de la edad y de los criterios de inclusión al programa. Métodos: Estudio observacional prospectivo realizado en un centro de fertilidad privado. Se aplicó la técnica de la DTEC D3/D5-6 de acuerdo al protocolo del Centro Valenciano de Fertilidad y Esterilidad (CEVALFES) a 58 pacientes durante el período septiembre 2012-2014 y que obtuvieron dos o más embriones de buena calidad. Resultados: La tasa de éxito general fue de un 39,65 %. Estos resultados se compararon con las tasas de éxito de la aplicación de la TE de D3 y de D5-6 de un solo embrión y de dos y tres embriones llevadas a cabo en un solo momento. La tasa de éxito general fue superior a las obtenidas con las TE D3 dobles y triples (29,23 % y 31,01 % respectivamente) y a las TE de blastocistos desvitrificados únicas y dobles (15,38 % y 15,15 % respectivamente). Conclusiones: La doble transferencia embrionaria consecutiva D3/D5-6 se perfila como una técnica eficaz para tomar la decisión de cuántos embriones transferir y el momento de hacerlo cuando la pareja cuenta con dos o más embriones de D3. Ello se traduce en una alternativa que puede mejorar los resultados de las técnicas de reproducción asistida en la actualidad.

Objective: The aim of this study was to evaluate a double consecutive embryo transfer day 3/ day 5-6 as an alternative to improve pregnancy rates among patients undergoing an IVF/ET program who obtain more than two day-3 embryos, without taking into account the number of previous attempts, age or IVF/ET program inclusion criteria. Method: Prospective observational study at a private fertility center. The double consecutive embryo transfer day 3/ day 5-6 approach was performed, according to the protocol outlined at CEVALFES, on 58 patients of our fertility center who obtained two or more good quality embryos between September 2012 and September 2014. Results: The overall pregnancy rate was 39,65%. These results were compared with single embryo transfers day 3 and day 5-6 and double and triple embryo transfers performed either on day 3 or on day 5-6. This overall pregnancy rate was higher than the rates obtained for double and triple day-3 ET (29,23% and 31,01% respectively) and for single and double thawed blastocyst ET (15,38% and 15,15% respectively). Conclusions: The double consecutive embryo transfer day 3 and day 5-6 approach presents itself as an effective alternative to inform decision making when two or more day-3 embryos are available: how many day-3 embryos are to be transferred and if ET can be performed consecutively on day 3 and day-5-6. This, in turn, becomes a choice that can improve current pregnancy rates.

A nonsurgical embryo transfer technique in mice / 生物工程学报

Although efficient nonsurgical transfer of embryos in mice would provide many advantages over a surgical method, the low success rate of nonsurgical transfer has hampered its acceptance and use. Here, a plastic catheter was used to mimic embryo transfer process and then the transfer efficiency was evaluated by intrauterine trypan blue dye dispersion. Also 3.5-day blastocysts from natural pregnant mice were transferred through cervix into uterine horns. The results show that 70.9% of CD-1 mouse 3.5-day blastocysts transferred into unilateral uterine horns of pseudopregnant 2.5-day recipients can be developed to live newborns, and an efficient mouse nonsurgical embryo transfer technique was established. The technique was simple, rapid, inexpensive, unlikely to get contaminated, ethical and do not need specialized apparatus, and can completely replace surgical embryo transfer techniques. Moreover, the mouse nonsurgical embryo transfer technique provides a research model for human and other large animal embryo transfer.

Pregnancy rate and live birth rate from in vitro fertilization treatment / Монголын Анагаах Ухаан

BackgroundConducting IVF treatment in laboratory environment by using ARTechnologies in idiopathic infertility,which cannot be treated by both drug treatment and surgical treatment in Mongolia, and preparinglegal environment are the crucial issues in treating infertility. The study is theoretically and practicallysignificant to properly determine pregnancy rate and live birth rate after embryo transfer treatmentin Mongolia.GoalAim of the study is to determine pregnancy rate and live birth rate after IVF treatment in coupleshaving infertility problem.Objectives1. To determine pregnancy rate in conjunction with age factor after conducting In vitro fertilizationin laboratory environment in couples with infertility problem.2. To determine live birth rate after pregnancy in conjunction with age factor.Materials and MethodsThe information of 180 couples aged 22-46 y.o., who received IVF treatment in “Unimed International”hospital’s IVF Center between March 2014 and June 2015, has been used in this study. Pregnancyrate as well as live birth rate after in vitro fertilization treatment has been determined. Statistic datawere calculated by SPSS-20 and Microsoft office excel 2013.ResultsThe average age of 180 women treated by In vitro fertilization treatment was 35.21±5.1 y.o. Sortedby age group: aged ≤30 y.o. was 34 (18.8%), aged between 31-34 y.o. was 41 (22.7%), agedbetween 35-39 y.o. was 80 (44.4%) and aged ≥40 y.o. was 25 (13.8%). 71 (39.44%) of womentreated by In vitro fertilization became pregnant. Sorted by their age: aged ≤30 y.o. was 18 (52.9%),aged between 31-34 y.o. was 20 (48.7%), aged between 35-39 y.o. was 28 (35.0%) and aged ≥40y.o. was 5 (20.0%). Resulted live birth from pregnancy is 56 (78.8%). Sorted by their age: aged≤30 y.o. was 16 (88.8%), aged between 31-34 y.o. was 18 (90.0 %), aged between 35-39 y.o. was22 (78.5%) and aged ≥40 y.o. was 2 (40.0%). Multiple births from total live birth is 16 (28.57%),amongst them 14 (25.0%) were twin births and 2 (3.51%) of them had triplet birth.Conclusions:1. IVF treatment in laboratory environment by using ARTechnologies in idiopathic infertility, whichcannot be treated by both drug treatment and surgical treatment, have good results (averagepregnancy rate was 39.44%).2. Live birth rate from total pregnancy was 56 (78.8%).3. Multiple birth from total live birth is 16 (28.57%). 14 (25.0%) were twins and 2 (3.51%) weretriplets.4. The IVF success rate depends on variable factors such as maternal age, cause of infertility,embryo status, reproductive history and lifestyle factors.

Impact of different mouse blastocyst donors on the efficiency of germline chimera production from C57BL/6 embryonic stem cells / 中国实验动物学报

Objective To improve the gene targeting efficiency with C57BL/6 embryonic stem ( ES) cells.Meth-ods Three different genetically modified C57BL/6 ES cell lines, named TLX3, Ai3K and SL, were microinjected into ICR, B6( Cg)-Tyrc-2J and BALB/c mouse blastocysts, respectively.The efficiency was statistically evaluated according to three aspects:blastocyst collection, chimera production and germline transmission.Results None of the three ES cell lines was germline transmitted with B6(Cg)-Tyrc-2J mice as blastocyst donors, while it was achieved with both BALB/c and ICR mouse blastocysts.Compared in the aspect of blastocysts collection, ICR mouse was much better than BALB/c mouse (P<0.05), and the chimera production efficiency of ICR mouse was comparable to that of BALB/c mouse (P =0.115). As to the germline transmission efficiency, that of BALB/c mice is significantly higher than that of the ICR mice ( P<0.01).Conclusions The germline transmission efficiency of BALB/c mouse is highest among these three mouse strains. However, it has the disadvantages of blastocyst collection, developmental delay and zona pellucida fragility, compared with ICR mouse.Therefore, ICR mouse is also a good candidate as blastocyst donor for embryonic stem cell microinjection.

Influence of L-arginine during bovine in vitro fertilization.

The objective of this work was to evaluate the effect of using L-arginine during in vitro fertilization (IVF) on in vitro embryonic development using Bos taurus and Bos indicus semen. Effect of different concentrations (0, 1, 10 and 50 mM) of L-arginine, added to the IVF medium, was evaluated on the fertilization rate at 18 h post-fertilization (hpf), NO3-/NO2- production during IVF by the Griess colorimetric method (30 hpf), cleavage and blastocyst rates (on Day 2 and Day 7 of culture, respectively) and total blastocyst cell number (Day 7 of culture). The results reveal that the addition of 50 mM L-arginine to IVF medium, with either Bos taurus or Bos indicus spermatozoa, decreased the cleavage rate and blastocyst rate compared to the control group. Other concentrations did not affect embryo production. However, 1 mM L-arginine with Bos indicus semen increased the proportion of hatched blastocysts. These results indicate that high L-arginine concentrations may exhibit toxic effects on bovine gametes during in vitro fertilization.

Embryonic stem-like cells derived from in vitro produced bovine blastocysts

The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like) cells from the inner cell mass (ICM) of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35 percent) ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF). Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

Study on the Development of Efficient Vitrification of Human Blastocysts / 대한불임학회지

OBJECTIVE: The purpose of this study was to evaluate the survival rate of vitrified blastocyst according to the freezing vessels, equilibration time in cryoprotectant and artificial dehydration method. METHODS: Human blastocysts were vitrified after loading onto the plastic straw, open-pulled straw (OPS), electron microscopy grid (EM grid) for 1.5 min or 3 min. They also were directly plunged into LN2 within 30sec. For artificial shrinkage of blastocysts, 36 gauge fine needle was pushed at the cellular junction of the trophectoderm into the blstocoele cavity until it shrank without damage of inner cell mass. RESULTS: The survival rate of vitrified blastocysts on plastic straw, OPS, EM grid as freezing vessels were 26.7, 13.0 and 60.5%, respectively. The survival rate of EM grid was significantly higher than that of plastic straw and OPS (p<0.05). For 1.5 min equilibrium, the survival rates of early blastocyst (EB), middle blastocyst (MB) and late blastocyst (LB) were 64.4, 81.0, and 20.0% respectively. For 3 min equilibrium, the survival rates of EB, MB, and LB were 69.9, 50.0 and 57.5% respectively. The survival rates of EB and MB were significantly higher than that of LB in 1.5 min equilibrium group (p<0.05), however, the significance was not observed in 3 min equilibrium groups. In cytoplasmic shrinkage before vitrification, the survival rates of EB, MB and LB were 92.9, 100 and 75.9% respectively. The survival rate of MB was significantly higher than that of LB (p<0.05). The survival rates of vitrified blastocysts by artificial dehydration and slow-frozen blastocysts were not significantly different as 88.9 and 66.7%, respectively. CONCLUSION: This study showed that the vitrification of human blastocysts using EM grid and artificial dehydration is an effective method. Therefore, these methods would be an useful techniques for blastocyst cryopreservation.

Effect of different exposure periods to the infrared 1.48 m diode laser on the inner cell mass and trophectoderm of blastocysts.

This study was carried out to determine the effect of the duration of exposure to an infrared 1.48 mm diode laser, on the number of cells in the inner cell mass and the trophectoderm of blastocysts following laser-assisted embryo biopsy. A total of 102 mouse embryos were used in the study. The embryos were randomly divided into three groups; group A (n = 22), group B (n = 47) and group C (n = 33). The embryos in group A were biopsied using the laser with an exposure of 600 ms, whereas those in group B were biopsied using the same laser with an exposure of 5 ms. The embryos in group C were incubated in culture without any procedures, as a control group. The blastocyst formation rates of group B (46/47, 97.8%) and group C (33/33, 100%) were significantly higher than that of group A (12/22, 54.5%). The number of cells in the inner cell mass, trophectoderm and the total number of cells in the blastocyst in group A (16.1 ฑ 5.1, 35.5 ฑ 10.9, 51.6 ฑ 12.9) were similar to those in group B (14.0 ฑ 5.6, 36.0 ฑ 12.7, 50.0 ฑ 18.3). The number of cells in the inner cell mass, trophectoderm and the total number of cells in the blastocysts in group C (19.1 ฑ 6.5, 45.8 ฑ 14.0, 65.0 ฑ 18.7) was significantly higher than those of the study groups. In conclusion, the longer duration of exposure to the infrared 1.48 mm diode laser might adversely affect blastocyst formation. However, it might not affect the quality of the blastocysts with regard to the number of cells in the inner cell mass and the trophectoderm.

Establishment of Human Embryonic Stem Cells Derived from Frozen-Thawed Blastocysts / 대한불임학회지

OBJECTIVE: This study was to establish the human embryonic stem (ES) cells derived from frozenthawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. METHODS: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. RESULTS: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and beta-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. CONCLUSION: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.

Physiological aspects of blastocyst uterine interaction.

An interaction between the blastocyst and the uterus is essential for establishment of pregnancy. Because maternal estrogen is not an absolute requirement, estrogen of embryonic origin has been implicated in this process in the pig and the rabbit. Furthermore, estrogen forming capacity has been documented in the blastocyst of these species. However, while the complete machinery for steroid synthesis in the pig balstocyst has been demonstrated, the issue is still unresolved for the rabbit blastocyst. In the present communication we have shown that 17α-hydroxylase and C17-20-lyase, enzymes involved in the formation of androgens (C19-steroids) from C21-steroids (progestins), are present in day-6 rabbit blastocysts. C17-20-lyase activity was undetectable to low in day-5 and increased in day-6 balstocysts. The activity was further increased in day-6 blastocysts cultured for 24 h. Because prostaglandins have been implicated in uterine vascular changes at about the time of implantation and pregnancy establishment, and because catechol estrogens are more potent than phenolic estrogens in stimulating prostaglandin synthesis in the blastocyst and the uterus, we determined catechol estrogen forming capacity in the rabbit and pig blastocyst. Catechol estrogen forming capacity (estrogen-2/4-hydroxylase) in the pig blastocyst appears on day 10 of pregnancy, peaks on day 12 and then declines. Our preliminary experiments also indicate that day-6 rabbit blastocysts have catechol estrogen forming capacity. On the basis of our present findings and of others, we propose that catechol estrogens of embryonic origin mediate the stimulatory effect of estrogens on prostaglandin synthesis in the embryo and/or the uterus and thus participate in the process of establishment of pregnancy.