Institute for Reproductive Medicine and Science (IRMS) at Saint Barnabas, Livingston, NJ, USA.
Department of Genetics, Rutgers University, Piscataway, NJ, USA.
J Assist Reprod Genet. 2024 May;41(5):1341-1356. doi: 10.1007/s10815-024-03064-2. Epub 2024 Mar 4.
PURPOSE/STUDY QUESTION: Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence?
Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes).
Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1).
The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing. Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.
目的/研究问题：ICSI 过程中卵母细胞透明带的穿刺是否允许周围两性离子缓冲液流入人卵母细胞，并导致胚胎发育能力改变？
本研究使用经伦理委员会批准的人类卵母细胞，通过共聚焦荧光显微镜（n=80 个人类 MII 卵母细胞）确定膜穿刺后周围缓冲液无限制流入卵母细胞的情况，以及使用超低输入 RNA 测序（n=40 个人类 MII 卵母细胞）确定 HEPES、MOPS 和碳酸氢盐缓冲液对卵母细胞转录组的选择缓冲液流入的影响。
在假 ICSI 过程中穿刺人类 MII 卵母细胞的透明带会导致周围培养缓冲液无限制地流入卵母细胞，这是技术员无法控制的。转录组分析显示，在穿刺缓冲液组中细胞骨架转录本明显减少，在碳酸氢盐缓冲液组中胚胎能力转录本（IGF2 和 G6PD）水平较高，在 HEPES 和 MOPS 组中应激诱导转录抑制因子转录本（MAF1）水平较高，在 HEPES 缓冲液组中许多染色体维持转录本（SMC3）水平降低。HEPES 缓冲液组还显示出更高水平的转录本，表明氧化（GPX1）和溶酶体应激（LAMP1）增加。
两性离子缓冲液对内在细胞机制的影响引起了人们对其在 IVF 临床应用中的广泛关注。主要关注点是 ICSI 程序，在此过程中，膜穿刺后允许周围缓冲液流入卵母细胞。选择生理碳酸氢盐缓冲液可能会减少对卵母细胞的施加压力，从而改善胚胎发育和临床结果，因为细胞内 MOPS，尤其是 HEPES，可能会通过转录组变化对内在生物学机制产生负面影响。这些发现进一步支持在 ICSI 过程中使用碳酸氢盐缓冲液作为卵母细胞保持介质。
