RESUMO
Objective To investigate the real correlation between embryo density and developmental outcome of cleavage embryos on day 3 by adjusting covariates. Methods Data of 1196 embryos from 206 couples undergoing in vitro fertilization and embryo transfer(IVF-ET) treatment were retrospectively analyzed. Embryos were hypoxia-cultivated in a fixed 30 μl microdrop in culture dishes. Embryo quality on day 3 was evaluated and proportion of good quality cleavage embryos on day 3 was used as the endpoint. Maternal age, paternal age, antral follicles, level of anti-mullerian hormone (AMH), type of infertility, controlled ovarian stimulation(COS) protocol, length of stimulation, number of retrieved oocytes, and type of insemination were chosen as covariates. Results A total of 1196 embryos were included and analyzed. Three embryo densities were routinely used: 30 μl/ embryo [1 embryo/ (30 μl·drop)], 15 μl/ embryo [2 embryos / (30 μl·drop)] and 10 μl/ embryo [3 embryos/ (30 μl·drop)]. The number of embryos assigned into these three groups were 434, 646 and 116 embryos separately. The average proportion of good quality embryos on day 3 in 10 μl/ embryo group were lower than that in both 15 and 30 μl/ embryo group (29. 31% vs 40. 25%,P 0. 05). In the regression analysis, without adjusting any covariables, the good quality embryos rate on day 3 of the 10 μl/ embryo group was 43% lower than that of the 30 μl/ embryo group (0. 57, 95% CI 0. 32, 1. 03), and there was no statistical difference (P>0. 05). In the minimum-adjusted model 2 (adjusted the level of AMH and type of insemination), proportion of good quality embryos on day 3 in group of 10 μl/ embryo significantly decreased by 51% (0. 49, 95% CI 0. 27,0. 90, P < 0. 05) compared with that in group of 30 μl/ embryo. Conclusion In a 30 μl microdrop, compared with individual cul turing, group culturing with the density of 10 μl / embryo did not benefit the development of the cleavage embryos and 30 μl / embryo was the optimal embryo density.
RESUMO
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)