title: A simple and effective method for the isolation of inner cell mass samples from human blastocysts for gene expression analysis.
abstract: The isolation of pure inner cell mass (ICM) and trophectoderm (TE) cells from a single human blastocyst is necessary to obtain accurate gene expression patterns of these cells, which will aid in the understanding of the primary steps of embryo differentiation. However, previously developed pure ICM isolation methods are either time-consuming or alter the normal gene expression patterns of these cells. Here, we demonstrate a simple and effective method of ICM samples isolation from human blastocysts. In total, 35 human blastocysts of all stages with expanded and good morphology were incubated in calcium/magnesium-free HEPES medium for 5 min before micromanipulation. With the aid of a laser, a biopsy pipette was inserted directly into the blastocoel for the suction-based removal of ICM samples. The ICM samples were obtained through simple mechanical pulling force or laser assistance, and each isolation process required 3-4 min. The isolated ICM and TE fractions were subjected to single-cell real-time quantitative RT-PCR to evaluate keratin 18 (KRT18) expression. Finally, 33 paired ICM and TE samples were verified using gene expression analysis. KRT18 was readily detectable in all TE cells but absent in 30 ICM counterparts, indicating a pure ICM isolation rate of 90.9% (30/33). The relative KRT18 expression of three TE samples compared with their three contaminated ICM counterparts was 19-fold (P < 0.001), indicating that the contamination was very weak. These results demonstrate that our ICM isolation method is simple and effective.
authors.affiliation: Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-Sen University, 58 Zhongshan Road II, 510080, Guangzhou, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Yanwen
authors.initials: Y
authors.lastname: Xu
authors.forename: Chenhui
authors.initials: C
authors.lastname: Ding
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Canquan
authors.initials: C
authors.lastname: Zhou
mesh_terms: D053624:Blastocyst Inner Cell Mass / Q000166:cytology*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D008846:Micromanipulation / Q000379:methods*; D059010:Single-Cell Analysis