Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 200011, Shanghai, China.
Department of Assisted Reproduction, First Maternity and Infant Hospital, Tongji University School of Medicine, 201204, Shanghai, China.
Reprod Biol Endocrinol. 2021 Nov 4;19(1):163. doi: 10.1186/s12958-021-00845-7.
The application of artificial oocyte activation (AOA) after intracytoplasmic sperm injection (ICSI) is successful in mitigating fertilization failure problems in assisted reproductive technology (ART). Nevertheless, there is no relevant study to investigate whether AOA procedures increase developmental risk by disturbing subsequent gene expression at different embryonic development stages.
We used a mouse model to explore the influence of AOA treatment on pre- and post-implantation events. Firstly, the developmental potential of embryos with or without AOA treatment were assessed by the rates of fertilization and blastocyst formation. Secondly, transcriptome high-throughput sequencing was performed among the three groups (ICSI, ICSI-AOA and dICSI-AOA groups). The hierarchical clustering and Principal Component Analysis (PCA) analysis were used. Subsequently, Igf2r/Airn methylation analysis were detected using methylation-specific PCR sequencing following bisulfite treatment. Finally, birth rate and birth weight were examined following mouse embryo transfer.
The rates of fertilization and blastocyst formation were significantly lower in oocyte activation-deficient sperm injection group (dICSI group) when compared with the ICSI group (30.8 % vs. 84.4 %, 10.0 % vs. 41.5 %). There were 133 differentially expressed genes (DEGs) between the ICSI-AOA group and ICSI group, and 266 DEGs between the dICSI-AOA group and ICSI group. In addition, the imprinted gene, Igf2r is up regulated in AOA treatment group compared to control group. The Igf2r/Airn imprinted expression model demonstrates that AOA treatment stimulates maternal allele-specific mehtylation spreads at differentially methylated region 2, followed by the initiation of paternal imprinted Airn long non-coding (lnc) RNA, resulting in the up regulated expression of Igf2r. Furthermore, the birth weight of newborn mice originating from AOA group was significantly lower compared to that of ICSI group. The pups born following AOA treatment did not show any other abnormalities during early development. All offspring mated successfully with fertile controls.
AOA treatment affects imprinted gene Igf2r expression and mehtylation states in mouse pre- and post-implantation embryo, which is regulated by the imprinted Airn. Nevertheless, no significant differences were found in post-natal growth of the pups in the present study. It is hoped that this study could provide valuable insights of AOA technology in assisted reproduction biology.
在辅助生殖技术（ART）中，应用人工卵母细胞激活（AOA）来缓解卵胞浆内单精子注射（ICSI）后的受精失败问题是成功的。然而，目前尚无相关研究探讨 AOA 程序是否会通过干扰不同胚胎发育阶段的后续基因表达而增加发育风险。
我们使用小鼠模型来探讨 AOA 处理对植入前和植入后事件的影响。首先，通过受精率和囊胚形成率评估有无 AOA 处理的胚胎的发育潜力。其次，在 ICSI、ICSI-AOA 和 dICSI-AOA 组之间进行转录组高通量测序。采用层次聚类和主成分分析（PCA）进行分析。随后，采用亚硫酸氢盐处理后的甲基化特异性 PCR 测序检测 Igf2r/Airn 甲基化分析。最后，通过小鼠胚胎移植检测出生率和出生体重。
与 ICSI 组相比，卵母细胞激活缺陷精子注射组（dICSI 组）的受精率和囊胚形成率明显较低（30.8%比 84.4%，10.0%比 41.5%）。ICSI-AOA 组与 ICSI 组之间有 133 个差异表达基因（DEGs），dICSI-AOA 组与 ICSI 组之间有 266 个 DEGs。此外，印迹基因 Igf2r 在 AOA 处理组中上调，与对照组相比。AOA 处理组的 Igf2r/Airn 印迹表达模式表明，AOA 处理刺激母源等位基因特异性甲基化在差异甲基化区域 2 上扩展，随后启动父源印迹 Airn 长非编码（lnc）RNA，导致 Igf2r 的上调表达。此外，源自 AOA 组的新生小鼠的出生体重明显低于 ICSI 组。接受 AOA 处理的幼鼠在早期发育过程中没有表现出任何其他异常。所有后代均与可育对照成功交配。
AOA 处理影响小鼠植入前和植入后胚胎的印迹基因 Igf2r 表达和甲基化状态，该状态受印迹基因 Airn 调节。然而，在本研究中，没有发现幼鼠在出生后的生长过程中有显著差异。希望本研究能为辅助生殖生物学中的 AOA 技术提供有价值的见解。
