Abstract
Co-culturing with the cumulus and granulosa cells (CGC) provide a microenvironment which can ensure the normal growth and development of human pre-implantation embryos in vitro . Examining the quantitative and qualitative compositions of this environment is of an important value to produce the systems to culture human embryos in vitro . The paper described the human embryos of the fifth day of development (blastocyst stage), monolayer culture of cryopreserved and freshly isolated CGC. We investigated the influence of culturing on monolayer of freshly isolated and cryopreserved CGC on morphokinetic characteristics of human pre-implantation embryos obtained in vitro as well as the amino acid profile of the culturing media. It has been found that cryopreservation does not affect the ability of CGC to support the development of pre-implantation embryos and improves their quality. There was determined the difference in amino acid composition of the standard medium of those for co-culturing on monolayer of either native or cryopreserved culture and cumulus granulosa. The presence of CGC changes the biochemical profile of culturing medium by increasing the content of the amino acids such as tryptophan, proline, valine, ornithine and glutamine. Probl Cryobiol Cryomed 2016; 26(2):124-132.
Highlights
Materials and methods5,2 ± 0,9 dance with the European protocol on embryo protection [20] and a decision of Середня кількість фолікулів на пацієнтку, абс. од
Co-culturing with the cumulus and granulosa cells (CGC) provide a microenvironment which can ensure the normal growth and development of human pre-implantation embryos in vitro
Arginine, methionine, valine and leucine have been shown to be involved into the development of human embryos [8], increasing the concentration of amino acids during co-culturing on either fresh or cryopreserved CGC positively influenced the development of embryos and their quality improves
Summary
5,2 ± 0,9 dance with the European protocol on embryo protection [20] and a decision of Середня кількість фолікулів на пацієнтку, абс. од. Removal of oocyte-crown-cumulus complexes, assessment of oocyte maturity, in vitro fertilization and Матеріали та методи culturing of embryos were performed by the standard. The cells лексів, оцінку зрілості яйцеклітин, запліднення in were placed in a well and 1 ml culture medium was vitro та культивування ембріонів проводили за layered under mineral oil (Cook, Australia). Після виділення ооцитів фолікулярну device (CryoLogic CL 8800i, Australia) using a progрідину поміщали в конічні центрифужні пробірки і ram: cooling rate of 0.3 deg/min from 25°C down to залишали за кімнатної температури на годину. Клітини ресуспендували в 3 мл культу- cooled from –6 down to –35°C with the rate of рального середовища «Sydney IVF Fertilization 1 deg/min, afterwards they were immersed into liquid проблемы криобиологии и криомедицины problems of cryobiology and cryomedicine том/volume 26, No/issue 2, 2016. Культивування КГК проводили у СО2-інкубаторі («Sanyo 5MO», Японія) при 37°С, вологості [7]
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