Validation of blastocyst biopsy and next generation sequencing (NGS) for the purpose of preimplantation genetic screening (PGS)
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The aim of this study was to examine the temporal sensitivity of bovine embryos to culture environment after fertilization to determine which period, if any, is most critical in determining blastocyst quality. Bovine zygotes produced in vitro were divided into six groups and cultured either in vitro (in synthetic oviductal fluid, SOF), in vivo (in the ewe oviduct) or in a combination of both systems. Development to the blastocyst stage, the ability of the blastocysts to withstand cryopreservation and the relative abundance of several gene transcripts were examined. Culture in SOF for either 2 or 4 days, followed by subsequent culture in the ewe oviduct, resulted in a significantly lower yield of blastocysts than did all other methods, the effect being most marked in embryos that were cultured in SOF for 4 days. In contrast, culture in vivo for the first 2 or 4 days after fertilization followed by culture in vitro did not have such a marked effect on blastocyst development. Blastocysts produced after culture in the oviduct for 6 days had the highest rates of survival over 72 h after warming (100% survival at 24 h; >95% survival at 72 h). The embryos that spent the last 4 days of culture in vivo also had relatively high rates of survival (100% at 24 h, 73.7% at 72 h). Blastocysts produced entirely in SOF had very low rates of survival after vitrification, with <40% viable at 24 h and <20% survival at 72 h. Blastocysts derived from embryos that spent the first 2 days in vivo and the last 4 days in vitro had the lowest rates of survival (6.7%), whereas those that spent the last 2 days only in SOF had intermediate rates of survival (40.6%). These differences were reflected in the relative abundance of transcripts for the Bax gene.
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The aim of this experiment was to examine the survival of porcine embryos following exposure or vitrification in EFS solution. The work was carried out on cultured and non-cultured expanded and hatched blastocysts. The viability of treated embryos was assessed by their ability to develop in in vitro culture. The results showed a higher viability of cultured expanded blastocyst (35.1 percent) vitrified in EFS medium compared to those non-cultured (12.5 percent). The proportion of cultured hatched blastocyst vitrified in this medium was 13.2 percent, while none of the non-cultured hatched blastocyst survived vitrification. It is concluded that: 1. The EFS solution was more toxic to hatched blastocyst than expanded blastocyst 2. The expanded blastocyst was a more suitable developmental stage for vitrification than hatched blastocyst. Moreover, we demonstrated that the survival rate of vitrified cultured pig blastocyst was higher compared to non-cultured.
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The three different development stages of pronuclear(n=220),2-4 cell (n=227) and morula(n=127) were recovered.For each stage,half of the embryos were cultured to the blastocyst and frozen thereafter,while the remainder as control group was frozen just recovery and cultured to the blastocyst;the experimental group of the blastocyst was obtained from uterus and frozen immediately after recovery.The effects on mouse embryos from in vitro culture of early stage to blastocyst stage and on their ability to surviving from cyropreservation were assessed.The survival and birth rate of blastocyst derived from in vitro culture of pronuclear,2-4 cell,morula and blastocyst of in vivo were 56.4%,72.2%,81.6%,95.1% and 38.1%,41.6%,56.8%,74.3%,respectively.The survival and birth rate of pronuclear and 2-4 cell were significantly lower than the control group(P0.01,P0.05),which suggests that the viability of early embryos is affected by in vitro culture.The rate of blastocyst derived from in vitro culture of pronuclear and 2-4 cell after thawing is lower than their counterpart(P0.05);the results indicate that the in vitro culture of pronuclear and 2-4 cell of mouse to the blastocyst stage prior to be frozen improves their survival after thawing.
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