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.

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

Pregnancy rates and luteal phase characteristics of bovine embryo recipients treated with flunixin meglumine / Taxas de prenhez e características da fase lútea de receptoras de embriões tratadas com flunixina meglumina em bovinos

The aim of this study was to evaluate the effects of flunixin meglumine administration on pregnancy rates and luteal phase characteristics in bovine embryo recipients at the moment of embryo transfer. In experiment 1, in vitro produced embryos were transferred to 184 females divided as control and treated group (recipients treated with 1.1mg/kg flunixin meglumine). In experiment 2, 22 females were divided as control group; group 2 (animals submitted to a reproductive tract manipulation similar to an embryo transfer on the 7th day after estrous); and group 3 (females submitted to a manipulation and treatment with 1.1mg/kg flunixin meglumine). In experiment 1 no difference was observed between control and treated groups (40.2% and 44.6%, respectively) for pregnancy rates. In experiment 2 no difference was observed on the length of luteal phase between groups, however, animals in group 2 presented lower plasma progesterone concentrations than the control group and group 3. Therefore, we concluded that although the administration of flunixin meglumine at the moment of embryo transfer inhibited the reduction plasma progesterone concentrations, it was not effective in increasing pregnancy rates of bovine recipients.(AU)

O objetivo deste estudo foi avaliar os efeitos da administração de flunixina meglumina sobre as taxas de prenhez e características da fase lútea da receptora no momento da transferência de embriões em bovinos. No experimento 1, embriões produzidos in vitro foram transferidos para 184 fêmeas, divididas em grupos controle e tratado (tratados com 1,1mg/kg de flunixina meglumina). No experimento 2, 22 fêmeas foram divididas em grupo controle (n=7); grupo 2 (n=8; animais submetidos à manipulação do trato reprodutivo semelhante à transferência de embriões no sétimo dia pós-cio); e grupo 3 (n=7; fêmeas submetidas à manipulação e ao tratamento com 1,1mg/kg de flunixina meglumina). No experimento 1, não foi observada diferença nos grupos controle e tratado (40,2% e 44,6%, respectivamente) para as taxas de prenhez. No experimento 2, não houve diferença na extensão da fase lútea entre os grupos, entretanto os animais do grupo 2 apresentaram concentrações plasmáticas de progesterona mais baixas que o grupo controle e o grupo 3. Portanto, conclui-se que a administração de flunixina meglumina no momento da transferência de embriões inibiu a redução das concentrações plasmáticas de progesterona, no entanto não foi eficaz para aumentar as taxas de prenhez de receptoras em bovinos.(AU)

Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro / Atividade antioxidante de óleo essencial obtido de Lippia origanoides melhora a qualidade de embriões bovinos produzidos in vitro

The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50µM/mL Cysteamine; T3)2.5µg/mL; T4)5.0µg/mL and T5)10.0µg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.(AU)

O objetivo desse estudo foi avaliar a suplementação de meio de cultura de embriões com antioxidante obtido do extrato oleoso da Lippia origanoides no desenvolvimento e na qualidade de blastocistos produzidos in vitro. Oócitos coletados de ovários de matadouros foram maturados e fertilizados in vitro segundo procedimento laboratorial padrão. Zigotos foram cultivados em meio SOF suplementado de acordo com os seguintes tratamentos: T1) meio de cultivo embrionário sem suplementação antioxidantes; T2) 50µM/mL Cisteamina; T3) 2,5µg/mL; T4) 5,0µg/mL e T5) 10,0µg/mL do antioxidante obtido do extrato oleoso de Lippia origanoides. No sétimo dia de cultivo, os blastocistos foram fixados e avaliados para taxa de apoptose, número total de células e massa celular interna através do teste TUNEL. O uso de antioxidantes durante cultivo não aumentou (P>0,05) a taxa de produção final de blastócitos. Os tratamentos T2, T3, T4 e T5 tiverem menor índice apoptótico (p>0,05 - 4,5±1,1%, 8,4±2,5%, 3,4±1,1% e 5,5±0,9%, respectivamente) quando comparados a T2 (10,0±1,4%). O valor de massa celular interna não diferenciou (p>0,05) entre embriões de diferentes tratamentos. A adição de antioxidante obtido do extrato oleoso de Lippia origanoides reduziu a taxa de apoptose e melhorou a qualidade sem aumentar a produção in vitro de embriões bovinos.(AU)

Light-induced injury in mouse embryos revealed by single-cell RNA sequencing

BACKGROUND: Light exposure is a common stress factor in in vitro manipulation of embryos in the reproductive center. Many studies have shown the deleterious effects of high-intensity light exposure in different animal embryos. However, no transcriptomic studies have explored the light-induced injury and response in preimplantation embryos. RESULTS: Here, we adopt different time-courses and illumination intensities to treat mouse embryos at the 2-cell stage and evaluate their effects on blastulation. Meanwhile, single-cell transcriptomes from the 2-cell to blastocyst stage were analyzed after high-intensity light exposure. These data show that cells at each embryonic stage can be categorized into different light conditions. Further analyses of differentially expressed genes and GO terms revealed the light-induced injury as well as the potential repair response after high-intensity lighting. Maternal-to-zygote transition is also affected by the failure to remove maternal RNAs and deactivate zygotic genome expression. CONCLUSION: Our work revealed an integrated response to high-intensity lighting, involving morphological changes, long-lasting injury effects, and intracellular damage repair mechanisms.

Effect of the air filtration system replacement on embryo quality in the assisted reproduction laboratory / Efeito da troca do sistema de filtragem do ar na qualidade embrionária no laboratório de reprodução assistida

Abstract Improving infrastructural conditions of the in vitro fertilization laboratory, such as the air quality, has profound positive effects on embryo culture. Poor environmental conditions reduce the rate of embryo formation and, therefore, of pregnancy. This review article presents important publications regarding the impact of air quality in human reproduction laboratories on embryo quality, pregnancy success, and live births. The studies demonstrate that the replacing the air filtration system improves significantly the environmental air quality, and, consequently, improves laboratory parameters, such as the fertilization rate, the number of blastocysts, the embryo implantation rate, and the number of live births. On the other hand, improving air quality decreases the number of abortions. Therefore, environmental parameters that improve embryo quality and increase healthy child birth ratesmust be themain targets for the assisted reproduction laboratory quality control.

Resumo Melhorar as condições de infraestrutura do laboratório de fertilização in vitro, com influência na qualidade do ar, tem efeitos positivos profundos na qualidade do embrião. As más condições ambientais do ar reduzem a taxa de sucesso na formação de embriões e a taxa de gravidez. Este artigo de revisão apresenta importantes publicações sobre o impacto da qualidade do ar dentro do laboratório de reprodução humana na qualidade do embrião, no sucesso de gravidez e no número de nascidos vivos. Os estudos demonstram que a troca do sistema de filtração de ar melhora significativamente a qualidade do ar ambiente, e consequentemente, melhora os parâmetros laboratoriais, tais como a taxa de fertilização, o número de blastocistos, a taxa de implantação e o número de nascidos vivos. Por outro lado,amelhora da qualidade do ar diminui o número de abortos. Portanto, os parâmetros ambientais que melhoram a qualidade do embrião e aumentam as taxas de nascimentos de crianças saudáveis devem ser os principais alvos para o controle de qualidade do laboratório de reprodução assistida.

Effective individual culture system for in vitro production of bovine embryos / Sistema eficaz de cultivo individual de embriões bovinos produzidos in vitro

A new and effective protocol to culture bovine embryos without coculture and with individualized culture media has been established, which would allow the study of a single embryo's metabolism. For this purpose, bovine embryos were produced in vitro by standard protocols in three different types of media: KSOM, SOFaa, and KSOM followed by SOFaa at day 2. Presumptive zygotes were divided into six groups: control, cultured in groups (C-KSOM, C-SOFaa, and C-KS), and individual well system (W-KSOM, W-SOFaa, and W-KS). Cleavage and blastocyst rates were assessed on days 2 and 7 respectively. Relative quantification of transcripts related to important metabolic processes (GLUT1, GLUT3, GSK3, SOD1, HSPD1, G6PD) were assessed in C-KS and W-KS blastocysts. Results show that cleavage was significantly higher only in W-KSOM when compared to C-KSOM, while blastocyst rates differ only between C-SOF and W-SOF. All the other comparisons did not present statistical difference. Moreover, gene expression analysis revealed that blastocysts cultured in groups and in the individual well system present similar transcription patterns. Thus, the obtained conclusion was that the individual well system performed could be used as an effective alternative protocol for individual culture of bovine embryos, since the rates are similar to routine group culture.(AU)

Estabeleceu-se um protocolo novo e eficaz de cultivo individual de embriões bovinos sem o uso de cocultivo e sem compartilhamento de meio visando à análise do metabolismo individual do embrião. Para isso, embriões foram produzidos in vitro por protocolos convencionais em três diferentes tipos de meio: KSOM, SOFaa e KSOM seguido por SOFaa no dia 2. Os zigotos presumíveis foram divididos em seis grupos: controles (cultivo em grupo ­ C-KSOM, C-SOFaa e C-KS) e sistema de poços individuais (W-KSOM, W-SOFaa e W-KS). As taxas de clivagem foram avaliadas nos dias 2 e 7, respectivamente. Além disso, a quantificação relativa de transcritos relacionados a importantes processos metabólicos (GLUT1, GLUT3, GSK3, SOD1, HSPD1 e G6PD) foi avaliada nos blastocistos dos grupos C-KS e W-KS. Os resultados mostram que as taxas de clivagem foram maiores apenas no grupo W-KSOM quando comparado ao grupo C-KSOM, enquanto a taxa de blastocistos diferiu apenas entre os grupos C e W-SOF. Além disso, a análise da expressão gênica mostrou que blastocistos cultivados em grupo ou em sistema de poços individuais são semelhantes quanto à expressão gênica. Assim, a conclusão obtida foi que o sistema individual proposto pode ser utilizado como um protocolo alternativo eficiente para o cultivo individual de embriões de bovino, uma vez que suas características permanecem semelhantes àquelas do sistema convencional de produção de embriões.(AU)

Adição da proteína específica do oviduto de porcas (pOSP) e da melatonina em meios de maturação e o efeito na clivagem in vitro de embriões suínos / Addition of the oviduct specific protein of porcine (pOSP) and melatonin in maturation media and their effect on the in vitro production of pig embryos

No presente estudo, utilizou-se a melatonina e a proteína específica do oviduto (pOSP) nos meios de maturação in vitro. Foram avaliadas a expansão do complexo cumulus-ovócito (CCOs), as concentrações intracelulares de espécies reativas de oxigênio (ROS) e o desenvolvimento embrionário nos diferentes grupos (C = controle; T1 = somente com melatonina; T2 = com melatonina e pOSP e T3 somente com pOSP). No tocante à expansão do CCOs, houve diferença (P<0,05) dos valores obtidos no grupo C em relação aos valores médios dos grupos T1, T2 e T3, porém não houve diferença entre os valores obtidos nos tratamentos (P>0,05). Na dosagem de ROS, não houve diferença entre os valores médios obtidos no grupo C (26,4±10,9) e o valor verificado no grupo T1 (23,4±7,8), porém no grupo T2 (21,3±9,7) o valor médio mostrou-se satisfatório em relação ao valor do grupo C. No entanto, o valor médio do grupo T3 (16,6±10,5) foi o que demonstrou resultado mais satisfatório quando comparado aos demais grupos (P<0,05). A produção de embriões foi avaliada por meio da taxa de clivagem. Não houve diferença (P >0,05) entre os valores obtidos entre o grupo C (48,9 %) e os valores verificados nos grupos T1 (51,5 %), T2 (50 %), T3 (57,7 %), nem destes entre si. Este estudo permitiu concluir que a proteína específica do oviduto recombinante e a melatonina foram eficientes em melhorar a expansão dos CCOs. Além disso, as células tratadas com pOSP mostraram-se com menor quantidade de ROS, podendo a pOSP ser considerada um antioxidante proteico.(AU)

The present study used melatonin and recombinant oviduct specific protein (pOSP) in in vitro maturation medium (IVM). The expansion of the cumulus-oocyte complexes (COCs), the intracellular concentrations of reactive oxygen species (ROS) and embryo development of the different groups were evaluated (C = control; T1 = melatonin; T2 = melatonin and pOSP and T3 = pOSP). Regarding the COCs expansion, the groups T1, T2 and T3 showed satisfactory results compared with group C (P<0.05), but there was no difference between treatments (P>0.05). In the ROS dosage, there was no difference between the mean values obtained in group C (26.4 ± 10.9) and group 1 (23.4 ± 7.8). However, in group 2 (21.3 ± 9.7), the average value was found to be satisfactory in relation group C. Despite that, the average value of treatment 3 (16.6 ± 10.5) was the most satisfactory result found compared to the other groups (P<0.05). The production of embryos was evaluated by cleavage rate, there was no difference between the values obtained in group C and the values recorded in groups T1 (51.5 %), T2 (50 %), T3 (57.7 %), and among them. This study showed that the pOSP and the melatonin were effective in the improvement of the expansion of COCs cells. In addition, the cells that were treated with pOSP presented a lower amount of ROS, allowing the pOSP to be considered a proteic antioxidant.(AU)

Valoración de la adopción de embriones humanos congelados desde el punto de vista de la filosofía moral, la ética laica y dos religiones monoteístas / Valuating frozen human embryo adoption from a moral philosophical point of view, lay ethics and two monotheist religions / Valoração da adoção de embriões humanos congelados do ponto de vista da filosofia moral, a ética laica e duas religiões monoteístas

La búsqueda de la eficacia en la fecundación in vitro hace que se produzcan más embriones que los que se implantarán, lo que produce un excedente de embriones, que es congelado. Esto hace que ineludiblemente el número de embriones humanos congelados aumente. Entre las soluciones para dichos embriones humanos congelados está la donación/adopción de los mismos. Ineludiblemente esta práctica conlleva objetivos problemas éticos. En este trabajo se evalúa la eticidad de la donación/adopción de embriones humanos congelados desde la perspectiva de la filosofía moral, lo que podríamos llamar una "ética laica" y dos de las religiones monoteístas: la musulmana y la judía.

The search for IVF efficacy leads to a higher embryo production than it is necessary for implantation; this results in an excess of embryos which are kept frozen. This amount of frozen embryos inevitably increases. The donation/adoption are among the possible solutions for these frozen embryos. However, this practice has objective ethical problems. This article considers the ethical aspects of the donation / adoption of frozen human embryos from the point of view of moral philosophy, from what we could call "secular ethics" and from two monotheistic religions: Muslim and Jewish.

A busca da eficácia na fecundação in vitro faz com que se produzam mais embriões dos que se implantarão, o que produz um excedente de embriões, que é congelado. Isto faz com que inquestionavelmente o número de embriões humanos congelados aumente. Entre as soluções para os ditos embriões humanos congelados está na doação/adoção dos mesmos. Ineludivelmente esta prática implica objetivos problemas éticos. Neste trabalho se avalia a eticidade da doação/adoção de embriões humanos congelados a partir da perspectiva da filosofia moral, o que poderíamos chamar uma "ética laica" e duas religiões monoteistas: a mulçumana e a judia.

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.

In vitro development and chimeric efficiency of mouse-porcine interspecies chimeric embryos in different culture systems / 生物工程学报

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human's, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.

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.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos / Suplementação com soro fetal bovino altera a modificação de histona H3R26me2 durante o período pré-implantacional em embriões bovinos produzidos in vitro

In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation...

A produção in vitro (PIV) de embriões de bovinos não é apenas de grande importância econômica para a pecuária, mas é também um importante modelo para estudar o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a modificação de histona, H3R26me2 durante o desenvolvimento pré-implantacional em embriões bovinos produzidos in vitro, cultivados com ou sem suplementação de soro fetal bovino (SFB), bem como comparar essa modificação específica entre mórulas produzidas in vitro e in vivo. Após a maturação in vitro e fertilização, embriões foram cultivados com suplementação de 0 ou 2,5% SFB. O desenvolvimento embrionário foi avaliado e embriões foram coletados e fixados em diferentes fases durante o desenvolvimento (2, 4, 8 e 16 células, mórula e blastocisto). Os embriões fixados foram avaliados por imunofluorescência utilizando um anticorpo para H3R26me2. Imagens de embriões corados foram analisadas baseadas na porcentagem do DNA total. Embriões cultivados com 2,5% SFB tiveram uma taxa de desenvolvimento ao estágio de blastocisto maior que o grupo que não recebeu suplementação com SFB (34.85±5,43% vs 23.38±,93%; P<0,05). Níveis de H3R26me2 variaram para ambos os grupos ao longo do desenvolvimento. No grupo 0% SFB, a marcação para H3R26me2 foi mais intensa nos estágios de 4 células (P<0,05), 16 células (P<0,05) e mórula (P<0.05). No grupo 2.5% SFB, apenas os embriões de 4 células tiveram marcação significativamente maior que todas as outras fases (P<0,01). Mórulas produzidas in vivo apresentaram níveis de H3R26me2 semelhantes ao grupo 0% SFB, e ambos foram significativamente maiores que o grupo 2.5% SFB. Estes resultados sugerem que a modificação de histona H3R26me2 é regulada durante o desenvolvimento pré-implantacional de embriões bovinos, e que as condições de cultura alteram de maneira importante esta regulação...

Evaluation of a father and son with atypical chronic myeloid leukemia with SETBP1 mutations and a review of the literature

We report the case of a father and son diagnosed with atypical chronic myeloid leukemia (aCML). Both patients harbored SETBP1 mutations, which are present in 24.3% of aCML patients. Moreover, both shared the variant encoding p.Pro737His, but the aCML severity was greater in the son because of the presence of two other missense mutations causing p.Asp868Asn and p.Ser885Arg alterations. SETBP1 mutations may be associated with an adverse prognosis, so their detection would help in the diagnosis of aCML and the determination of a patient's prognosis.

In vitro fertilization (IVF) in mammals: epigenetic and developmental alterations: scientific and bioethical implications for IVF in humans

The advent of in vitro fertilization (IVF) in animals and humans implies an extraordinary change in the environment where the beginning of a new organism takes place. In mammals fertilization occurs in the maternal oviduct, where there are unique conditions for guaranteeing the encounter of the gametes and the first stages of development of the embryo and thus its future. During this period a major epigenetic reprogramming takes place that is crucial for the normal fate of the embryo. This epigenetic reprogramming is very vulnerable to changes in environmental conditions such as the ones implied in IVF, including in vitro culture, nutrition, light, temperature, oxygen tension, embryo-maternal signaling, and the general absence of protection against foreign elements that could affect the stability of this process. The objective of this review is to update the impact of the various conditions inherent in the use of IVF on the epigenetic profile and outcomes of mammalian embryos, including superovulation, IVF technique, embryo culture and manipulation and absence of embryo-maternal signaling. It also covers the possible transgenerational inheritance of the epigenetic alterations associated with assisted reproductive technologies (ART), including its phenotypic consequences as is in the case of the large offspring syndrome (LOS). Finally, the important scientific and bioethical implications of the results found in animals are discussed in terms of the ART in humans.

In vitro development of canine somatic cell nuclear transfer embryos in different culture media

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

Migração espermática em suínos após inseminação artificial intrauterina profunda / Sperm migration in pigs after deep intrauterine artificial insemination

A inseminação artificial intrauterina profunda (IIP) é de grande importância para a indústria suinícola, em função do maior número de doses produzidas por reprodutores de alto mérito genético e da possibilidade da utilização de biotecnologias, como sêmen sexado e/ou congelado. Entretanto, necessita-se compreender com maior propriedade os mecanismos pelos quais os espermatozoides colonizam as tubas uterinas. Assim sendo, pretende-se com o presente experimento avaliar a existência ou não de migração intraperitoneal de espermatozoides inseminados profundamente em um dos cornos uterinos, mediante a obtenção de oócitos fertilizados no corno contralateral à inseminação e seccionado na base, na junção com o corpo do útero. Quatorze fêmeas pluríparas foram divididas em dois grupos experimentais, sendo que em um deles as fêmeas foram submetidas à secção da base de um dos cornos uterinos (Grupo Operado, n = 7), enquanto as do Grupo Controle (n = 7) não foram submetidas a nenhuma intervenção cirúrgica. Ambos os grupos foram submetidos à IIP, sendo as fêmeas abatidas 5±1,2 dias após a última inseminação. Os sistemas genitais das fêmeas foram coletados, dissecados e o número de corpos lúteos contados em ambos os ovários. A recuperação dos embriões foi feita por meio de lavagem das tubas e cornos uterinos com solução de PBS (Phosphate Buffered Saline), após o que se avaliou os fluidos coletados em lupa para a identificação de embriões. Em ambos os grupos experimentais, foram encontrados embriões nos segmentos do sistema genital de ambos os lados. Apenas uma fêmea apresentou embriões nos segmentos em somente um dos lados no grupo operado. Diante dos resultados aqui observados, concluiu-se que a migração espermática no suíno pode ocorrer tanto por via retrógrada pelo útero quanto por migração intraperitoneal. Estes achados certamente contribuirão para aumentar a eficiência da técnica de IIP, sendo de grande valia para o aprimoramento da indústria suinícola...

Deep intrauterine insemination (DUI) is of great importance for the swine industry as it can increase the efficiency in the use of boars of high genetic merit, and facilitate the use of biotechnologies such as frozen and sexed semen. However, a better understanding of the mechanisms by which the sperm colonize the uterine tubes is essential. The aim of the present study was to investigate the existence of intrauterine sperm migration after DUI in one uterine horn, through the fertilization of oocytes in the contra lateral uterine horn. Fourteen multiparous sows were divided into two experimental groups: Operated (n = 7), where females had a segment close to the base of the uterine horn surgically removed, and Control (n = 7), females with intact uterus. Both groups were inseminated through DUI and slaughtered 5±1.2 days after the last insemination. The reproductive tracts collected were dissected and the number of corpora lutea counted in both ovaries. Embryo recovery was performed though flushings of uterine tubes and horns with Phosphate Buffered Saline solution and further examination under a dissecting microscope. Embryos were found in the uterine horns of both sides of the reproductive tract in both experimental groups. In the operated group, just one female had embryos in only one side of the reproductive tract. The results presented herein suggest that sperm migration in pigs may occur both in a retrograde way through the uterus and by intraperitoneal migration. These findings will certainly contribute to increase the efficiency of the DUP technique, which is of great importance for the improvement of the swine industry...

Effect of leptin on in vitro nuclear maturation and apoptosis of buffalo [bubalus bubalis] oocyte

Leptin, as a 16 kDa adipokine, is a pleiotropic cytokine-like hormone that primarily secreted from adipose tissue. It also involves in the regulation of energy homeostasis, neuroendocrine function, immunity, lipid and glucose homeostasis, fatty acid oxidation, angiogenesis, puberty and reproduction. The aim of this study was to investigate the effects of in vitro addition of leptin to in vitro maturation [IVM] medium on buffalo oocyte maturation and apoptosis. In this experimental study, Ovaries from apparently normal reproductive organs of slaughtered adult buffaloes [Bubalus bubalis] with unknown breeding history were collected from Urmia Abattoir, Urmia, Iran, and were transported immediately to the laboratory in a thermos flask containing sterile normal saline with added antibiotics. Oocytes were aspirated from 2-8 mm visible follicles of the ovaries using an 18-G needle attached to a 10 ml syringe. IVM medium included tissue culture medium-199 [TCM-199], 10% fetal bovine serum [FBS], 22 microg/ml sodium pyruvate, 0.5 IU/ml ovine follicle-stimulating hormone [oFSH], 0.5 IU/ml ovine luteinizing hormone [oLH], 1 microg/ml oestradiol, 50 microg/ml gentamycin, and leptin [0 [control], 10, 50, and 100 ng/ml]. The good quality buffalo oocytes [batches of 10 oocytes] were placed in a culture plate containing six 50 microl droplets of maturation medium, covered with sterilized mineral oil, and then incubated at 38.5?C with 5% CO2 in air for 24 hours. The maturation of oocytes was evaluated under a stereomicroscope by detecting the first polar body extrusion of oocytes. FITC-Annexin V - propidium iodide [PI] staining method was used to detect oocyte apoptosis. From a total of 115 collected ovaries, 1100 oocytes were recovered among which 283 oocyte were suitable for IVM. In the groups of leptin treated with 0 [control], 10, 50 and 100 ng/ml, the percentage of oocytes maturation was 74.65, 83.81, 77.85, and 75.40%, while the percentage of oocytes apoptosis was 9.83, 9.54, 9.93, and 10.42%, respectively. Our results showed that addition of 10 ng/ml leptin to buffalo IVM medium increased oocyte maturation, significantly, as compared with that in control group. However, addition of leptin to IVM medium had no significant influence on buffalo oocyte apoptosis. Our findings suggested that addition of 10 ng/ml leptin to IVM medium of buffalo oocyte can improve oocyte nuclear maturation. Furthermore, we showed that there is no relation between in vitro addition of leptin to buffalo oocyte IVM medium and oocyte apoptosis

Embryotoxicity of Senecionis Scandentis Hebra on in vitro cultured mouse embryos / 药学学报

The purpose of this study is to evaluate the embryotoxicity of alkaloids in Senecionis Scandentis Hebra on in vitro cultured mouse embryos. Mouse whole embryo culture (WEC) was applied in this study. Post-implantation (8.5 d) mouse embryos were isolated from their mothers, and cultured in medium of immediately centrifuged serum (ICS) with different concentrations of seneciphylline (target concentrations were 100, 50, 25 and 12.5 μg x mL(-1)) or senkirkine (target concentrations were 50, 25 and 12.5 μg x mL(-1)) for 48 h. After culturing completed, the development and organic morphodifferentiation of the cultured embryos were evaluated microscopically. Treatment with seneciphylline and senkirkine had adverse effects on the development and organic morphodifferentiation of embryos. The effect also had clear dose-response. Alkaloidals in Senecionis Scandentis Hebra had embryotoxicity on cultured embryos, which indicated that pregnant people exposed to Senecionis Scandentis Hebra may get potential risk on fetus.

In vitro maturation, fertilization and embryo culture of oocytes obtained from vitrified auto-transplanted mouse ovary

The purpose of this study was to investigate the in vitro survival and developmental potential of oocytes obtained from vitrified mouse ovaries transplanted to a heterotopic site. In this experimental study, two-week-old mice were unilaterally ovariectomized after anesthesia. The ovaries were vitrified by cryotop. After two weeks, the ovaries were thawed and autotransplanted to the gluteus muscle tissue. Three weeks later the mice were killed, after which we removed and dissected the transplanted and opposite right ovaries. Cumulus oocyte complexes [COCs] and denuded oocytes were evaluated for in vitro maturation [IVM], in vitro fertilization [IVF] and in vitro development [IVD]. The control group consisted of sevenweek- old age-matched mice ovaries. All vitrified-transplanted [Vit-trans] ovaries contained some oocytes that survived. Following IVM, IVF and IVD, there were 41.7% out of 12 cultured zygotes that reached the 8-cell stage. Our experiment supports the progressive role of long-term graft survival after wholeovarian cryopreservation by vitrification and subsequent heterotopic transplantation. It is possible to recover viable follicles and oocytes that have the ability to develop in vitro