Evaluating histone modification analysis of individual preimplantation embryos.

BACKGROUND: We previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT). RESULTS: WOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing. CONCLUSIONS: These results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

Effects of tris (2-carboxyethyl) phosphine hydrochloride treatment on porcine oocyte in vitro maturation and subsequent in vitro fertilized embryo developmental capacity.

Oocyte in vitro maturation (IVM) is a crucial process that determines subsequent in vitro embryo production. The present study investigated the effects of the antioxidant tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCL) on the in vitro maturation of porcine oocytes and in vitro developmental competence of fertilized embryos. Oocytes were matured in IVM medium based on four concentration groups of TCEP-HCL (0, 50, 100, and 200 µM) treatment. 100 µM TCEP-HCL treatment significantly increased the oocyte first polar body extrusion rate, monospermy rate and subsequent in vitro fertilized embryo developmental capacity (cleavage rate, blastocyst formation rate, and blastocyst total cell number) compared to those in the control group. Furthermore, 100 µM TCEP-HCL treatment significantly reduced the levels of reactive oxygen species, significantly increased glutathione levels and mitochondrial content compared to those in the control group. Moreover, 100 µM TCEP-HCL treatment significantly decreased the oocyte apoptosis, blastocyst apoptosis compared to that in the controls. In summary, these results indicate that 100 µM TCEP-HCL treatment improves the quality and developmental capacity of in vitro-fertilized embryos by decreasing oxidative stress in porcine oocytes.