在体外受精(IVF)之前,生发囊泡完整卵母细胞的体外成熟(IVM)在动物中广泛实施。在人类辅助生殖中,通常保留用于保存生育力或禁止卵巢刺激的地方。标准实践包含复杂蛋白质(CP),以血清和/或白蛋白的形式,IVM培养基模拟卵巢卵泡环境。然而,CP的未定义性质,连同批次变异和关于其来源的伦理问题,需要开发更明确的配方。一种已知的卵泡液成分,褪黑激素,具有多方面的作用,包括代谢调节剂和抗氧化剂。在某些情况下,它可以促进卵母细胞成熟。在这个发展阶段,生发囊泡完整的卵母细胞容易发生非整倍性和表观遗传失调。
■为了确定发育,在牛IVM期间去除CP和包括褪黑激素的细胞遗传学和表观遗传学后果。
■该研究包括2×2阶乘排列,比较(i)包含或排除CP,和(ii)添加(100nM)或省略褪黑激素,在IVM期间。从刺激的周期中回收卵丘-卵母细胞复合物(COC)。在IVM和IVF之后,将推定的受精卵在标准培养基中培养至第8天。RNAseq在分离的卵丘细胞上进行,对分离的滋养外胚层细胞进行细胞遗传学分析(基于SNP的算法),和DNA甲基化分析(减少代表亚硫酸氢盐测序)对分离的细胞内细胞团。
■在IVM期间去除CP导致胚泡发育适度减少,而添加褪黑激素在有CP的情况下是有益的,但在没有CP的情况下是有害的。IVM培养基的组成不影响染色体异常的性质或发生率,但卵丘细胞转录本表达表明COC中代谢改变(主要是脂质)。这些作用先于几天后在扩大和孵化的胚泡中建立不同的代谢和表观遗传特征。
■这些发现突出了脂质的重要性,特别是甾醇,IVM期间COC的代谢。它们为未来的研究奠定了基础,这些研究寻求开发化学定义的IVM系统,以产生细胞遗传学和表观遗传学正常的可转移胚胎。
In vitro maturation (
IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into
IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation.
To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM.
The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during
IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following
IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass.
Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of
IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts.
These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.