Cell-free DNA in Embryo Culture Media as Non-invasive Biomarker of the Quality of Embryo Cleavage

Background: The success of in vitro-fertilization (IVF) cycles is determined in large part by the quality of embryo cleavage, which in turn, is dependent on the quality of the embryo culture media (CM). Many factors can influence the quality of embryo CM, one of which is the levels of Cell Free Deoxyribonucleic acid (DNA). Understanding the association between Cell-free DNA levels in embryo CM and the quality of embryo cleavage could help improve the quality of IVF techniques. Methods: This prospective study was conducted with 96 spent CM from patients undergoing IVF cycle, in order to determine relationships of Cell-free DNA levels in embryo CM with embryo cleavage quality on day 3. After intracytoplasmic sperm injection (ICSI), 48 embryos were evaluated on day 3 of their development, according to their cell number. Day 2 and day 3 CM corresponding to each one of the embryos was analyzed, by quantitative PCR, for estimation of Cell-free DNA levels. Results: The results revealed a significant increase in Cell-free DNA levels on day 2 CM corresponding to 4 to 6 cell embryos compared to those corresponding to 7 to 8 cell embryos (p=0.04). As for day 3 CM, the results showed no significant difference between the Cell-Free DNA levels in CM of 7-8 and those of 4-6 cell embryos (p=0.4). Also, cell free DNA levels in embryo CM, were significantly higher on day 2 compared to day 3 for both 7-8 and 4-6 cell embryos (p=0.03; p=0.04). Conclusion: We conclude that cell-free DNA levels in CM might be associated with delayed embryo cleavage.

Comparison of the Effects of GMCSF-Containing and Traditional Culture Media on Embryo Development and Pregnancy Success Rates

Abstract Objective The use of granulocyte macrophage colony-stimulating factor (GM-CSF)-containing medium, which is a commercial medium that is used for cultivation of embryos in in vitro fertilization (IVF) treatments, has been suggested to increase the efficiency of this procedure in patients with previous multiple unsuccessful attempts. In this retrospective study, we analyzed GM-CSF-containing embryo culture media compared with traditional culture media in terms of development of embryos, pregnancy, and ongoing pregnancy success and live birth rates. Methods This is a prospective case control study conducted in a single center. A total of 131 unexplained infertility patients were included in the study. A cohort of 69 patients whose embryos were cultured in GM-CSF-containing medium and a control group of 62 age-matched patients whose embryos were cultured in conventional Sage One Step medium were included in the study. The major study outcomes were achievement of pregnancy and ongoing pregnancy rate at 12 weeks of gestation. Results The pregnancy and ongoing pregnancy rates of the patients whose embryos were cultured in GM-CSF-containing medium were 39.13% and 36.23%, respectively. These were higher than the rates of the control group, which were 30.65% and 29.03%, respectively, although this difference was not statistically significant. In addition, the 5th-day embryo transfer percentage in the GM-CSF group was higher than in the control group (34.78% versus 27.4%). Conclusion The main findings of our study were that there was no difference between the GM-CSF-enhanced medium and the control group in terms of our major study outcomes. However, blastomere inequality rate and embryo fragmentation rates were lower in the GM-CSF group.

Asynchrony between in vivo and in vitro rabbit embryos

Background: Comparative features of embryos developed under in vitro and in vivo conditions are particularly important in designing embryo transfer procedures that fulfil embryo-recipient synchronization requirements. Objective: To determine the degree of asynchrony in rabbit embryo development between cultured and in vivo embryos. Methods: A total of 55 non- lactating multiparous female rabbits were used. Embryos were classified as 16-cells or early morulae at 48 hours post-coitum (hpc). Embryos were cultured during 30 or 32 h and embryo development was compared with in vivo embryos of 72 hpc. In vitro and in vivo embryos at 72 hpc were classified as early or compacted morulae. Bayesian statistics was used. Difference between in vivo and in vitro embryos and the actual probability of the difference between the in vivo and in vitro embryo higher than zero (P) was estimated. Results: The percentage of compacted morulae was higher in in vivo embryos than in in vitro embryos with +6 h of asynchrony (73.5 and 32.8%, P=1.00). But the percentage of compacted morulae was similar with +8 h asynchrony. Conclusions: In vitro embryos delay their development by + 8 hours compared to in vivo embryos.

Antecedentes: El desarrollo comparativo de embriones producidos in vitro e in vivo es particularmente importante para el diseño de procedimientos de transferencia de embriones cuando se requiere sincronización entre el embrión y la hembra receptora. Objetivo: Determinar el grado de asincronía en el desarrollo embrionario entre embriones in vivo y cultivados. Métodos: Un total de 55 conejas multiparas no lactantes fueron utilizadas. Los embriones se clasificaron en 16 células o mórulas tempranas a las 48 horas después del coito (hpc). Los embriones se cultivaron durante 30 ó 32 horas y el desarrollo embrionario se comparó con embriones de 72 hpc obtenidos in vivo. Los embriones in vitro e in vivo a 72 hpc se clasificaron como mórulas tempranas o compactas. Se utilizó estadística bayesiana. Se estimó la diferencia entre embriones in vivo e in vitro y la probabilidad de que la diferencia sea superior a cero (P). Resultados: El porcentaje de mórulas compactas fue mayor en embriones in vivo que en embriones in vitro con +6 horas de asincronía (73,5 y 32,8%, P=1,00), pero el porcentaje de mórulas compactas fue similar con asincronía de +8 horas. Conclusión: Los embriones cultivados retrasan +8 horas su desarrollo en comparación con los embriones in vivo.

Antecedentes: A aquisição do desenvolvimento de embriões produzidos in vitro e in vivo é particularmente importante na concepção de procedimentos de transferência de embriões em que a sincronização entre o embrião e a fêmea receptora é necessária. Objetivo: Determinar o grau de assincronia no desenvolvimento embrionário entre embriões cultivados e in vivo. Métodos: Um total de 55 coelhos multíparos não lactantes foram usados. Os embriões foram classificados em 16 células ou mórulas iniciais 48 horas de gestação (hpc). Os embriões foram cultivados por 30 ou 32 horas e o desenvolvimento embrionário foi comparado com embriões de 72 hpc obtidos in vivo. Embriões in vitro e in vivo a 72 hpc foram classificados como mórulas precoces ou compactadas. Estatísticas bayesianas foram usadas. A diferença entre embriões in vivo e in vitro e a probabilidade de que a diferença seja maior que zero (P) foi estimada. Resultados: A porcentagem de mórulas compactadas foi maior em embriões in vivo do que em embriões in vitro com +6 horas de assincronia (73,5 e 32,8%, P=1,00). Mas a porcentagem de mórulas compactadas foi semelhante com assincronia de +8 horas. Conclusão: Embriões cultivados atrasam seu desenvolvimento em +8 horas em comparação com embriões in vivo.

Well-of-the-well (WOW) versus polyester mesh (PM): a comparison of single-embryo culture systems in bovines

Background: Mexico is innovating in the livestock industry through in vitro generation of bovine embryos with technologies such as well-of-the-well (WOW) and polyester mesh (PM) single-embryo culture systems. These techniques allow to maintain embryos in separate areas of a shared culture medium. Objective: To compare the quantity and quality of bovine embryos produced in WOW and PM culture systems versus the conventional (CG) culture system. Methods: In total, 345 embryos fertilized in vitro were evaluated for blastocyst yield in the three culture systems. To count blastocyst cell numbers, 69 embryos in each system were differentially stained for trophectoderm (TE), inner cell mass (ICM), and apoptotic cells. A qPCR gene expression analysis was performed for embryos in all three systems. Results: The WOW, PM and CG systems developed similar amount of blastocysts (41, 35 and 36%, respectively; p>0.05). Blastocysts in all three systems showed adequate amounts of ICM and apoptotic cells. Blastocysts in the PM system showed a greater number of TE cells [63.7 versus 58.6% in the CG system (p0.05). The ATP5B expression was higher in WOW than in PM (p0.05). The TJP3 expression was higher in PM than in WOW and CG (p<0.05). Expression of ID2 and CLDN4 was higher in WOW than in PM and CG (p<0.05). The biplot graphic from Principal Component Analysis (PCA) revealed that CG was located near degenerated embryos, whereas PM was located near arrested embryos, larger ICM and TE, and TJP3 expression. The WOW was located toward blastocysts, morulae, and expression of CLDN4, ID2 and GNAS. Conclusion: Compared with CG, both the PM and WOW systems are good options for culturing single embryos in the bovine model. Moreover, the PCA results suggest that embryos developed in the WOW system have greater capacity for generating blastocysts with increased ability to form TE and ICM layers, which might improve implantation.

Antecedentes: México está innovando en la industria ganadera a través de la generación in vitro de embriones bovinos con tecnologías de cultivo individual como lo son Pozo dentro de Pozo (WOW) y Malla de Poliéster (PM). Estos mantienen los embriones en áreas separadas mientras comparten un mismo medio de cultivo celular. Objetivo: Comparar la cantidad y calidad de embriones bovinos producidos en los sistemas WOW y PM contra el sistema de cultivo convencional en grupo (CG). Métodos: En total se evaluaron 345 embriones fertilizados in vitro para determinar la producción de blastocistos generados en los tres sistemas. Para contar el número de células por blastocisto, 69 embriones en cada sistema se tiñeron diferencialmente para trofectodermo (TE), masa celular interna (ICM) y células apoptóticas. Se realizó un análisis de expresión génica por qPCR de los embriones obtenidos en los tres sistemas. Resultados: Los sistemas WOW, PM y CG desarrollaron similares cantidades de blastocistos (41, 35 y 36%, respectivamente; p>0,05). Los blastocistos en los tres sistemas mostraron cantidades adecuadas de ICM y células apoptóticas. Los blastocistos en el sistema PM mostraron un mayor número de células TE [63,7% versus 58,6% en el sistema CG (p0,05). La expresión de ATP5B fue mayor en WOW que en PM (p<0,05), pero similar a CG (p<0,05). La expresión de TJP3 fue mayor en PM que en WOW y CG (p<0,05). La expresión de ID2 y CLDN4 fue mayor en WOW que en PM y CG (p<0,05). El gráfico de biplot del análisis de componentes principales reveló que CG se encontró cerca de embriones degenerados, mientras que PM se encontró cerca de embriones en arresto, ICM, TE, y TJP3. El WOW se localizó hacia blastocistos, mórulas y la expresión de CLDN4, ID2 y GNAS. Conclusión: En el modelo bovino los sistemas PM y WOW son buenas opciones para cultivar embriones individuales, ya que se obtienen resultados muy similares a los obtenidos con el sistema CG. Además, los resultados de PCA sugieren que los embriones individuales desarrollados en el sistema WOW generan blastocistos con mayor capacidad de formar TE e ICM, lo que podría mejorar su éxito de implantación.

Antecedentes: O México está inovando na indústria pecuária por meio da geração in vitro de embriões bovinos com tecnologias de cultura de embriões individuais, bem como em poço (WOW) e malha de poliéster (PM). Estes mantêm os embriões em áreas separadas, enquanto compartilham o mesmo meio de cultura de células. Objetivo: Comparar a quantidade e a qualidade de embriões bovinos produzidos nos sistemas de cultura WOW e PM com o sistema convencional de cultura em grupo (CG). Métodos: No total, 345 embriões fertilizados in vitro foram avaliados para determinar a produção de blastocistos gerados nos três sistemas. O número de células por blatocisto foi contado, 69 embriões em cada sistema foram diferencialmente corados para trofectoderme (TE), massa celular interna (ICM) e células apoptóticas. Uma análise de expressão gênica qPCR foi realizada para os embriões obtidos nos três sistemas. Resultados: Os sistemas WOW, PM e CG desenvolveram quantidades semelhantes de blastocistos (41, 35 e 36%, respectivamente; p>0,05). Os blastocistos nos três sistemas mostraram quantidades adequadas de ICM e células apoptóticas. Os blastocistos no sistema PM mostraram um número maior de células TE [63,7 versus 58,6% no sistema CG (p0,05). A expressão de ATP5B foi maior no WOW do que no PM (p<0,05), mas semelhante ao GC (p<0,05). A expressão de TJP3 foi maior no PM do que no WOW e CG (p<0,05). A expressão de ID2 e CLDN4 foi maior no WOW do que no PM e CG (p<0,05). O gráfico biplot da análise de componentes principais revelou que CG foi encontrado próximo a embriões degenerados, enquanto PM foi encontrado próximo a embriões presos, ICM, TE e TJP3. WOW foi encontrado para ter blastocistos, mórulas e a expressão de CLDN4, ID2 e GNAS. Conclusão: Em comparação com o CG, os sistemas PM e WOW são boas opções para a cultura de embriões individuais no modelo bovino. Além disso, os resultados da PCA sugerem que embriões individuais desenvolvidos no sistema WOW têm maior capacidade de desenvolver blastocistos com maior capacidade de formar as camadas TE e ICM, o que poderia melhorar seu sucesso de implantação.

Aplicación de diferentes metodologías en el mejoramiento de lenteja (Lens culinaris Medik) / Application of different methodologies in lentil (Lens culinaris Medik) breeding

RESUMEN La lenteja (Lens culinaris Medik.) es una especie diploide autógama (2n=2x=14) perteneciente a la familia Fabaceae. Es uno de los cultivos más antiguos que se conocen, con 8.000 a 9.000 años de historia, y se encuentra entre los primeros domesticados en Medio Oriente. Las semillas tienen un alto valor nutricional. Este cultivo es un interesante sustituto del trigo en las rotaciones de cereales, pero su importancia es baja debido a la falta de buenas variedades con adaptación local. Uno de los principales problemas que enfrentan los mejoradores en nuestro país es la estrecha base genética del germoplasma cultivado y su bajo potencial de rendimiento. En 2004 se inició un programa de mejoramiento de lentejas para desarrollar nuevas variedades con adaptación a las condiciones predominantes en las áreas de cultivo de Argentina. El germoplasma se obtuvo del ICARDA (Centro Internacional de Investigación Agrícola en las Zonas Áridas) y de productores locales. Se utilizan métodos convencionales de mejoramiento basados en la hibridación y selección. Se han obtenido dos nuevas variedades, una del tipo macrosperma (Boyerito FCA) y la otra del tipo microsperma (Tacuarita FCA) mediante la aplicación de selección masal en poblaciones F2 provenientes de la hibridación de materiales seleccionados. Este programa complementa los métodos de mejora tradicional con técnicas biotecnológicas como la transgénesis, el uso de marcadores moleculares, el cultivo de embriones in vitro combinado con el método SSD para acortar el ciclo generacional, y el fenotipado digital.

ABSTRACT Lentil (Lens culinaris Medik.) is a self-pollinating diploid (2n=2x=14) species belonging to the Fabaceae family. It is one of the oldest crops known, with 8,000 to 9,000 years of history and it is among the earliest domesticates from the Near East Fertile Crescent. The seeds have high nutritional value. This crop is an interesting substitute to wheat in cereal rotations but its importance is low due to a lack of suitable varieties with local adaptation. Some of the major problems that Argentinian lentil breeders face are the narrow genetic base of the current cultivated germplasm and its low yield potential. A lentil breeding program was initiated in 2004 to develop new varieties with adaptation to prevalent conditions in growing areas of Argentina. Germplasm was obtained from ICARDA (International Center for Agricultural Research in the Dry Areas) and local producers. Conventional breeding methods using hybridization and selection are being carried out to develop improved varieties, broad the genetic base, and isolate superior recombinant inbred lines. Two new varieties have been obtained, one of the macrosperm type (Boyerito FCA) and the other of the microsperm type (Tacuarita FCA) through the application of mass selection in F2 populations from the cross of selected materials. This program complements traditional breeding methods with biotechnological techniques such as transgenesis, use of molecular markers, in vitro embryo culture combined with the SSD method to shorten the breeding time, and digital phenotyping.

Design and experiment of online monitoring system for long-term culture of embryo / 生物医学工程学杂志

In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for

Construction of microfluidics-based fallopian tube model for mimicking fertilization and early embryo culture in vivo / 中国组织工程研究

BACKGROUND: With the increasing proportion of infertility in the population, more and more attentions have been paid on assisted reproductive techniques. Fertilization and early embryo culture are the significant parts of assisted reproductive techniques; however, they remain unchanged in the last few decades. OBJECTIVE: To design a novel microfluidics-based fallopian tube model that can mimic the microenvironment of fertilization and early embryo culture in vivo. METHODS: Microfluidic device was manufactured by soft lithography method to mimic the anatomical characteristic of fallopian tube in vivo. Mouse oviduct primary epithelial cells were cultured and purified by explants culture method, and then the purified cells were identified by keratin immunofluorescence method. Epithelial cells were then loaded into the channel to mimic the biochemical environment of fallopian tube in vivo. The chip was connected to the automatic liquid changing device to mimic the liquid environment of fallopian tube in vivo. RESLUTS AND CONCLUSION: (1) The channel of this model is cylindrical with 2 cm of height and 1 cm of diameter, which were in accordance with the anatomical characteristic of the isthmus of fallopian tube in shape. (2) The keratin immunofluorescence was positive, which indicated that mouse oviduct primary epithelial cells can be obtained by explants culture method. (3) The cells were loaded into the channel to cover the wall of channel, which provided a biochemical microenvironment similar to that in vivo for fertilization and early embryo culture. After the chip was connected to the automatic liquid changing device, metabolic waste could be taken away and nutrient substance can be replenished in time, which mimics the real fluid environment in vivo. (4) This study combined microfluidics technology and assisted reproductive techniques to design a novel fallopian tube model, which mimics the micro-environment of fertilization and early embryo culture in vivo. This study has laid a foundation for further improvement of assisted reproductive techniques and the rate of fertilization and embryo optimization.

Outcome of modified intracytoplasmic sperm injection using zona pellucida -bound spermatozoa in male ;infertility / 中国基层医药

Objective To investigate the treatment outcome of modified intracytoplasmic sperm injection (ICSI)using zona pellucida(ZP)-bound sperm.Methods 82 patients with less,weak,abnormal sperm disease who were conformed to ICSI,were divided into traditional ICSI group and the group of modified ICSI using ZP-bound sperm according to ICSI case number.The results of normal fertilization rate,cleavage rate,high-quality embryo rate, planting rate,clinical pregnancy rate and early abortion rate were compared.Results The women's age,the sterility year,mature egg rate,normal fertilization rate,cleavage rate in the two groups had no statistically significant differ-ences (all P>0.05).The planting rate and clinical pregnancy rate of the observation group (46.6%,63.3%)were higher than those of the control group(38.5%,53.6%),but there were no statistically significant differences(all P>0.05).The using embryo rate and high-quality embryo rate of the observation group (73.9%,51.0%)were signifi-cantly higher than those of control group(65.8%,38.6%),the differences were statistically significant(χ2 =5.84,χ2 =11.6,all P<0.05).Conclusion Modified ICSI using ZP-bound sperm can effectively improve the embryos quality in ICSI.

Primer embarazo con transferencia de embriones seleccionados por morfocinética en el Perú / First pregnancy with transfer of morphokinetics-selected embryos in Peru

La selección embrionaria usando características morfológicas observadas por pocos segundos bajo el microscopio, ha sido la principal herramienta de selección en las técnicas de reproducción asistida. Sin embargo, el desarrollo embrionario es un proceso dinámico que con la introducción de incubadoras con microcámaras integradas, conocidas como incubadoras con sistema Time Lapse, ha permitido registrar eventos morfológicos y cinéticos del desarrollo embrionario que pueden ser útiles como marcadores de selección, denominándolos parámetros 'morfocinéticos'. En este reporte de caso damos a conocer el primer embarazo en el Perú mediante la transferencia de embriones seleccionados por parámetros morfocinéticos en una incubadora con sistema Time Lapse.

Embryo selection by using morphological characteristics has been the main tool to select the best embryo to transfer in assisted reproduction technology (ART). However, embryo development is a dynamic process that cannot be monitored with conventional microscopes. The introduction of incubators with an integrated micro-camera system, denominated time-lapse incubators, has allowed to register morphological and kinetic events in human embryos, be coming useful markers for embryo selection. In this report, we present the first pregnancy in Peru using morphokinetic parameters in a time lapse incubator.

Comparison of static culture, micro-vibration culture, and micro-vibration culture with co-culture in poor ovarian responders / 대한생식의학회지

OBJECTIVE: This study was conducted to compare the effects of static culture, dynamic culture, and the combination of dynamic culture with specialized surfaces involving co-culture on human embryonic development. Embryos cultured using conventional static culture (SC) techniques served as a control group. We compared dynamic culture using micro-vibration culture (MVC) and micro-vibration with co-culture (MCoC), in which autologous cumulus cells were used as a specialized surface. METHODS: We conducted a chart review of patients who were treated between January 2011 and November 2014 in order to compare embryonic development rates and pregnancy rates among the groups. Zygotes were cultured in micro-droplets, and embryos were subsequently selected for transfer. Some surplus embryos were cryopreserved, and the others were cultured for blastocyst development. A micro-vibrator was set at the frequency of 42 Hz for duration of 5 seconds per 60 minutes to facilitate embryo development. RESULTS: No significant differences among the groups were present in patient's characteristics. However, the clinical pregnancy rates were significantly higher in the MVC group and the MCoC group than in the SC group. No significant differences were found in the blastocyst development rate between the SC group and the MVC group, but the blastocyst development rate in the MCoC group was significantly higher than in the SC and MVC groups. CONCLUSION: The clinical pregnancy rate was significantly increased by the application of micro-vibration to the embryonic cultures of poor responders. The blastocyst development rate was significantly increased by the application of MCoC to surplus embryos.

Biocompatibility study of lithium disilicate and zirconium oxide ceramics for esthetic dental abutments

PURPOSE: The increasing demand for esthetically pleasing results has contributed to the use of ceramics for dental implant abutments. The aim of this study was to compare the biological response of epithelial tissue cultivated on lithium disilicate (LS₂) and zirconium oxide (ZrO₂) ceramics. Understanding the relevant physicochemical and mechanical properties of these ceramics will help identify the optimal material for facilitating gingival wound closure. METHODS: Both biomaterials were prepared with 2 different surface treatments: raw and polished. Their physicochemical characteristics were analyzed by contact angle measurements, scanning white-light interferometry, and scanning electron microscopy. An organotypic culture was then performed using a chicken epithelium model to simulate peri-implant soft tissue. We measured the contact angle, hydrophobicity, and roughness of the materials as well as the tissue behavior at their surfaces (cell migration and cell adhesion). RESULTS: The best cell migration was observed on ZrO₂ ceramic. Cell adhesion was also drastically lower on the polished ZrO₂ ceramic than on both the raw and polished LS2. Evaluating various surface topographies of LS2 showed that increasing surface roughness improved cell adhesion, leading to an increase of up to 13%. CONCLUSIONS: Our results demonstrate that a biomaterial, here LS2, can be modified using simple surface changes in order to finely modulate soft tissue adhesion. Strong adhesion at the abutment associated with weak migration assists in gingival wound healing. On the same material, polishing can reduce cell adhesion without drastically modifying cell migration. A comparison of LS2 and ZrO2 ceramic showed that LS2 was more conducive to creating varying tissue reactions. Our results can help dental surgeons to choose, especially for esthetic implant abutments, the most appropriate biomaterial as well as the most appropriate surface treatment to use in accordance with specific clinical dental applications.

Enhancement of germination, hyperhydricity control and in vitro shoot formation of Vasconcellea stipulata Badillo / Mejoramiento de la germinación, control de la hiperhidricidad y formación de brotes en Vasconcellea stipulata Badillo

Vasconcellea stipulata has great commercial importance because of its enzymatic activity and as a source for genetic improvement of papaya since it is resistant to the papaya ringspot virus. However, due to its low regeneration by seeds and limited knowledge of its genetic and pharmaceutical properties, this species is not widely cultivated. For propagation, in vitro culture of seeds has been used to address this problem, but hyperhydricity, a physiological disorder, mainly expressed in the developing embryonic axis and specifically associated with this species, is a significant constraint. In order to obtain elite material for culture of V. stipulata, the aim of this work was to increase germination, to control hyperhydricity in embryos and to evaluate the potential to induce morphogenic responses, i.e., shoot formation. Our results showed that it is possible to increase germination up to 53% under in vitro conditions within a short period in the presence of hydrogen peroxide. In addition, hyperhydricity was significantly reduced (50%) in vitro when gibberellic acid concentrations were included on a 1/2 Nitsch and Nitsch nutrient medium, resulting in approximately 80% recovery of viable seedlings. Finally, other plant growth regulators were evaluated and found to trigger shoot formation in axillary buds as well as induce the formation of callus in leaf sections derived of seedlings.

Vasconcellea stipulata posee una gran importancia comercial debido a su actividad enzimática y como fuente para el mejoramiento genético de papaya, debido a su resistencia al virus de la mancha anular de esta especie. Sin embargo, debido a su baja regeneración por semillas y al limitado conocimiento de sus propiedades genéticas y farmacéuticas, esta especie no es cultivada ampliamente. La propagación a través del cultivo in vitro de semillas se ha usado para contrarrestar este tipo de problema, pero la hiperhidricidad, un trastorno fisiológico, expresado principalmente en los ejes embrionarios en desarrollo y asociado específicamente a esta especie, es una restricción significativa. Con el fin de obtener material de élite para el cultivo de V. stipulata, el objetivo de este trabajo fue incrementar la germinación, controlar la hiperhidricidad en embriones y evaluar el potencial para inducir respuestas morfogénicas, es decir, la formación de brotes. Nuestros resultados mostraron que es posible aumentar la germinación hasta un 53% en condiciones in vitro, dentro de un período más corto en presencia de peróxido de hidrógeno. Además, la hiperhidricidad se redujo significativamente (50%) en condiciones in vitro cuando se incluyó ácido giberélico en bajas concentraciones en el medio 1/2 Nitsch y Nitsch. Esto permitió recuperar hasta aproximadamente el 80% de plántulas viables. Finalmente, otros reguladores de crecimiento vegetal evaluados, indujeron la formación de brotes en yemas axilares y la formación de callos en secciones de hoja derivadas de plántulas.

Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

Clinical outcomes of different transferring methods in patients who received different numbers of oocytes / 西安交通大学学报(医学版)

Objective To compare the clinical outcomes of two D3 embryo and single blastocyst transfer in patients retrieving different oocytes, so as to provide data support for selecting a clinical transfer strategy. Methods We made a retrospective analysis of patients who underwent in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI)between January and December 2014 in the Reproductive Medicine Center,the Third Affiliated Hospital of Zhengzhou University.The patients were divided into three groups according to the number of oocytes received:Group A (5-9 oocytes),Group B (10 - 14 oocytes)and Group C (≥ 1 5 oocytes).Patients in each group all received four different transfer methods as follows:transfer of two fresh D3 embryos (a ),transfer of one fresh blastocyst (b ),transfer of two D3 frozen embryos (c ),and transfer of one frozen blastocyst (d ).We compared the 2PN fertilization rate of oocytes,rate of available embryos and rate of good embryos among the three groups.We also compared the embryo implantation rate,biochemical pregnancy rate,clinical pregnancy rate, multiplets rate and abortion rate among the four transfer methods in each group.Results ① There were 667, 573,and 479 transfer cycles in Group A,Group B and Group C,respectively.The 2PN fertilization rate of IVF and available embryos rate was significantly higher in Group A than in Group B and Group C (P =0.003/P 0.05),but the implantation rate of c was significantly lower than that of a and d (P =0.027/0.020),d had a higher implantation rate than a and c in Group B (P =0.005/0.001).In Group C,the biochemical pregnancy rate and clinical pregnancy rate of d were significantly higher than those of a (P =0.048/0.027)and c (P =0.003/0.001).Patients in Group C also had a higher implantation rate than D3 embryos (P <0.05).③ The multiple pregnancy rate of single blastocyst transfer decreased compared with D3 embryos transfer in the three groups (P <0.05).Conclusion Single blastocyst transfer has both higher implantation rate and lower multiple pregnancy rate in high response patients (1 5 or more oocytes received).For patients who received 5-9 and 10-14 oocytes,D3 embryos have a similar clinical pregnancy rate with that of single blastocyst but a higher multiple pregnancy rate.Single vitrified-warmed blastocyst transfer has a higher clinical pregnancy rate.It is the best transfer method for patients who received more than 10 oocytes.

In vitro culture of embryos and establishment of embryonic stem cell lines / 中国组织工程研究

BACKGROUND:The successful establishment of human embryonic stem cel lines in vitro is of great significance to human embryonic development mechanism and developmental biology, cel and tissue transplantation in the treatment of certain diseases. OBJECTIVE:To summarize the progress of in vitro culture of embryos and establishment of embryonic stem cel lines, to explore the influential factors for in vitro culture of embryos, and the methods of culturing human discarded embryos, isolating inner cel mass and establishing embryonic stem cel lines, as wel as the establishing conditions for embryonic stem cel lines. METHODS:With the key words of“embryo, embryonic stem cel s, coculture, sequential culture”, the first author searched CNKI and SCI databases for literatures concerning in vitro culture and transplantation of embryos and establishment of embryonic stem cel lines published from 2000 to 2014. Systematic evaluation was conducted. Final y, 58 literatures were retained for result analysis. RESULTS AND CONCLUSION:The culturing condition for embryos in vitro is the key factor affecting embryo transfer outcomes, including culture medium component and culture system. In previous studies, the component and application of culture medium have changed greatly, and the culture system has altered from single culture to coculture and sequential culture. Ethical issues and embryonic origin restrictions restrict the establishment of human embryonic stem cel lines. Clinical y discarded low-quality embryos can be used as one of the material sources to establish human embryonic stem cel lines, which can effectively lessen the problem of embryo shortage during the establishment of human embryonic stem cel lines and reduce ethical disputes.

Comparison of teratogenecity induced by nano- and micro-sized particles of zinc oxide in cultured mouse embryos / 대한수의학회지

The increasing uses of zinc oxide nanoparticles (nZnO) in industrial and personal care products raise possible danger of using nZnO in human. To determine whether ZnO induces size-dependent anomalies during embryonic organogenesis, mouse embryos on embryonic day 8.5 were cultured for 2 days under 50, 100, and 150 microg of nZnO (< 100 nm) or micro-sized ZnO (mZnO; 80 +/- 25 microm), after which the morphological changes, cumulative quantity of Zn particles, and expressions of antioxidant and apoptotic genes were investigated. Although embryos exposed to 50 microg of ZnO exhibited no defects on organogenesis, embryos exposed to over 100 microg of ZnO showed increasing anomalies. Embryos treated with 150 microg of nZnO revealed significant changes in Zn absorption level and morphological parameters including yolk sac diameter, head length, flexion, hindbrain, forebrain, branchial bars, maxillary process, mandibular process, forelimb, and total score compared to the same dose of mZnO-treated embryos. Furthermore, CuZn-superoxide dismutase, cytoplasmic glutathione peroxidase (GPx) and phospholipid hydroperoxidase GPx mRNA levels were significantly decreased, but caspase-3 mRNA level was greatly increased in nZnO-treated embryos as compared to normal control embryos. These findings indicate that nZnO has severer teratogenic effects than mZnO in developing embryos.

In vitro culture of immature embryos of Cinnamomum tamala Nees.— The role of different factors.

Seed characteristics and in vitro culture of C. tamala embryos were studied. Embryos desiccated below 50% (fresh weight) exhibited poor morphogenetic response in vitro and confirmed the recalcitrant nature of seeds. The immature embryos of various developmental ages (4-16 week after flowering, WAF) were cultured on different strengths of MS medium. Morphogenesis responses were recorded after 10 days of culture. The best culture responses were achieved from the immature embryos of 12 WAF on MS medium with sucrose (3%, w/v), polyvinyl pyrollidone (100 mg L-1) and benzyl adenine (12 µM). Under optimum condition ~60% explants responded; and ~7.3 shoots buds developed per explants after 35 days of culture initiation. The shoot buds could be converted into micro-shoots on MS medium with sucrose (3%) and kinetin (3 µM). About 5.3 micro-shoots/shoot buds sprouted per sub-culture. The micro-shoots were rooted by maintaining them on MS medium with α-naphthalene acetic acid (3 µM) where within 6-8 wk of culture ~8-10 roots developed. The rooted plantlets were acclimatized in vitro before they were transferred to community potting mix and maintained in the poly-shade ca 75% shading. The transplants registered ~70% survival after two months of transfer.

An ideal oocyte activation protocol and embryo culture conditions for somatic cell nuclear transfer using sheep oocytes.

Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 μM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.

Comparison of the clinical outcomes of day 4 and 5 embryo transfer cycles / 대한생식의학회지

OBJECTIVE: The majority of embryo transfers (ETs) to date have been performed on day 3 to reduce the potential risk of developmental arrest of in vitro cultured embryos before ET. Development of sequential media has significantly improved culture conditions and allowed blastocyst transfer on day 5. While day 5 ET provides higher clinical pregnancy outcomes with reduced risks of multiple pregnancies, it still has potential risks of developmental arrest of IVF embryos. The aim of this study was to evaluate the clinical outcomes of day 4 ETs and compare the efficacy of day 4 ET with day 5 ET. METHODS: From 2006 to 2009, a total of 747 fresh IVF-ET cycles were retrospectively analyzed (day 4, n=440 or and day 5, n=307). The cycles with any genetic factors were excluded. The rates of matured oocytes, fertilization, good embryos, and clinical pregnancy of the two groups were compared. The chi-square test and t-test were used for statistical analysis. RESULTS: There were no significant differences between the two groups with respect to the mean age of the females and rates of matured oocytes. The pregnancy outcomes of day 4 ET (40.7%) were similar to those of day 5 ET (44.6%). The implantation rate of day 5 ET (24.2%) was significantly higher than that of day 4 ET (18.4%) (p=0.003). CONCLUSION: Day 4 ET can be chosen to avoid ET cancellation in day 5 ET resulting from suboptimal circumstances in the IVF laboratory, but the decremented quality of embryos for transfer and the decreased pregnancy rate must be taken into consideration.

In vitro rescue of interspecific embryos from Elaeis guineensis x E. oleifera (Arecaceae)

The African oil palm (Elaeis guineensis) is the most effective oil producer in tons per hectare. Nevertheless, its increasing cultivation in Latin America is harmed by the “lethal yellowing”. Genetic resistance to this anomaly can be found in the germplasm of American oil palm or caiaué (E. oleifera), a native species from the Amazon rainforest. However, the procedures adopted to induce seeds of E. guineensis to germination frequently result mild for interespecific hybrids. Embryo in vitro cultivation can be a viable option. This work was aimed initially to test liquid MS medium supplemented with different glucose or sucrose concentrations for the in vitro cultivation of zygotic embryos from E. guineensis x E. oleifera controlled pollinations. Additionally we investigated different compost mixtures to acclimatize the regenerated hybrid plantlets. Concentrations of 10, 20 and 30g/L of both sugars were tested on flasks containing five mature zygotic embryos, with 15 repetitions per treatment in a total of 450 explants. The number of embryos displaying shoots and radicles at least 2mm in length per experimental unit was evaluated during phase one of in vitro cultivation. Plantlets displaying shoots and radicles were transferred to phase two of in vitro cultivation and subsequently to acclimatization, under 70% shading with manual water supply. The experiments of acclimatization were conducted with 130 plantlets randomly distributed in pure horticultural compost, 3:1 or 1:1 compost:sand mixtures and each plantlet was defined as an experimental unit. Data were submitted to ANOVA, t test and analyzes of correlation (p≤0.05). Highest emergence rates were 97% for shoots and 73% for radicles, observed in MS medium supplemented with 20g/L (110mM) of glucose. This sugar in concentrations of 20 or 30g/L provided balanced shoot/root development, and this was considered one of the reasons for the higher frequency of plantlet establishment. The survival percentage was 55% after the first 43 days of acclimatization and by the fourth month, 66 plants developed simultaneously longer shoot and root systems in pure horticultural compost. in conclusion, radicle development was an impairment to plantlet establishment and was overcame under media with glucose above 110mM. Acclimatization could benefit from an extended period of in vitro development. Rev. Biol. Trop. 59 (3): 1081-1088. Epub 2011 September 01.

Elaeis guineensis es el productor de aceite más eficaz en toneladas por hectárea, su cultivo, cada vez mayor en América Latina, se ha visto perjudicado por el “amarilleamiento letal”. La resistencia genética a esta anomalía se puede encontrar en el germoplasma de la palma aceitera americana o caiaué (E. oleifera), una especie nativa de la selva amazónica. Sin embargo, los procedimientos adoptados para inducir la germinación de las semillas de E. guineensis frecuentemente produce resultados modestos para híbridos interespecíficos. El cultivo de embriones in vitro puede ser una opción viable. En este trabajo se probó el medio líquido MS complementado con diferentes concentraciones de glucosa o sacarosa en el cultivo in vitro de embriones cigóticos de E. guineensis x E. oleifera originados de polinización controlada. Además se investigaron diferentes mezclas de compost para aclimatar los híbridos regenerados. Las concentraciones de 10, 20 y 30 g/L de ambos azúcares se probaron en frascos que contenían cinco embriones cigóticos maduros, con 15 repeticiones por tratamiento y un total de 450 explantes. El número de embriones que muestran brotes y radículas de al menos 2mm de longitud por unidad experimental se evaluó durante la primera fase de cultivo in vitro. Las plántulas que mostraron brotes y radículas fueron trasladadas a la segunda fase de cultivo in vitro y, posteriormente, se aclimataron, por debajo de 70% de sombra con el suministro manual de agua. Los experimentos de aclimatación se llevaron a cabo con 130 plántulas distribuidas al azar en el compost hortícola puro, compost 3:1 o 1:1: mezclas de arena y cada plántula se definió como una unidad experimental. Los datos fueron sometidos a un análisis de varianza, prueba t y análisis de correlación (p≤0.05). Las tasas más altas de emergencia fueron 97% y 73% para brotes y radículas respectivamente, en el medio MS complementado con 20g/L (110mM) de glucosa. Este azúcar en concentraciones de 20 o 30g/L permitió un desarrollo balanceado de brotes/desarrollo de raíces, que fue considerado como una de las razones de la alta frecuencia de establecimiento de las plántulas. El porcentaje de supervivencia fue de un 55% después de los primeros 43 días de aclimatación y por el cuarto mes, 66 plantas desarrollaron simultáneamente hojas largas y un sistema radical en el compost hortícola puro. En conclusión, el desarrollo radicular fue un impedimento para el establecimiento de plántulas y se superó en el medio con glucosa por encima de 110mM. La aclimatación podría beneficiarse con un largo período de desarrollo in vitro.