Follicle Biology Laboratory (FOBI), UZ Brussel, Vrije Universiteit Brussel, Brussels, Belgium.
Animal Physiology, Institute of Agricultural Sciences, ETH Zurich, Switzerland.
Hum Reprod. 2019 Sep 29;34(9):1640-1649. doi: 10.1093/humrep/dez121.
Does imprinted DNA methylation or imprinted gene expression differ between human blastocysts from conventional ovarian stimulation (COS) and an optimized two-step IVM method (CAPA-IVM) in age-matched polycystic ovary syndrome (PCOS) patients?
No significant differences in imprinted DNA methylation and gene expression were detected between COS and CAPA-IVM blastocysts.
Animal models have revealed alterations in DNA methylation maintenance at imprinted germline differentially methylated regions (gDMRs) after use of ARTs. This effect increases as more ART interventions are applied to oocytes or embryos. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for patients. CAPA-IVM is an improved IVM system that includes a pre-maturation step (CAPA), followed by an IVM step, both in the presence of physiological compounds that promote oocyte developmental capacity.
STUDY DESIGN, SIZE, DURATION: For DNA methylation analysis 20 CAPA-IVM blastocysts were compared to 12 COS blastocysts. For RNA-Seq analysis a separate set of 15 CAPA-IVM blastocysts were compared to 5 COS blastocysts.
PARTICIPANTS/MATERIALS, SETTING, METHODS: COS embryos originated from 12 patients with PCOS (according to Rotterdam criteria) who underwent conventional ovarian stimulation. For CAPA-IVM 23 women were treated for 3-5 days with highly purified hMG (HP-hMG) and no hCG trigger was given before oocyte retrieval. Oocytes were first cultured in pre-maturation medium (CAPA for 24 h containing C-type natriuretic peptide), followed by an IVM step (30 h) in medium containing FSH and Amphiregulin. After ICSI, Day 5 or 6 embryos in both groups were vitrified and used for post-bisulphite adaptor tagging (PBAT) DNA methylation analysis or RNA-seq gene expression analysis of individual embryos. Data from specific genes and gDMRs were extracted from the PABT and RNA-seq datasets.
CAPA-IVM blastocysts showed similar rates of methylation and gene expression at gDMRs compared to COS embryos. In addition, expression of major epigenetic regulators was similar between the groups.
LIMITATIONS, REASONS FOR CAUTION: The embryos from the COS group were generated in a range of culture media. The CAPA-IVM embryos were all generated using the same sperm donor. The DNA methylation level of gDMRs in purely in vivo-derived human blastocysts is not known.
A follow-up of children born after CAPA-IVM is important as it is for other new ARTs, which are generally introduced into clinical practice without prior epigenetic safety studies on human blastocysts. CAPA-IVM opens new perspectives for patient-friendly ART in PCOS.
STUDY FUNDING/COMPETING INTEREST(S): IVM research at the Vrije Universiteit Brussel has been supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680), the Fund for Research Flanders (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen-FWO-AL 679 project, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69Ref Nr 2016-119) and the Vrije Universiteit Brussel (IOF Project 4R-ART Nr 2042). Work in G.K.'s laboratory is supported by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council. The authors have no conflicts of interest.
在年龄匹配的多囊卵巢综合征（PCOS）患者中，常规卵巢刺激（COS）和优化的两步体外成熟（CAPA-IVM）方法中，囊胚的印迹 DNA 甲基化或印迹基因表达是否存在差异？
在 COS 和 CAPA-IVM 囊胚中，未检测到印迹 DNA 甲基化和基因表达的显著差异。
动物模型显示，在使用辅助生殖技术（ART）后，印迹生殖系差异甲基化区（gDMR）的 DNA 甲基化维持发生改变。这种影响随着对卵母细胞或胚胎进行更多的 ART 干预而增加。IVM 是一种低刺激的 ART，具有减少激素相关副作用和降低患者风险的特点。CAPA-IVM 是一种改良的 IVM 系统，包括预成熟阶段（CAPA），随后是 IVM 阶段，均在促进卵母细胞发育能力的生理化合物存在下进行。
研究设计、规模、持续时间：对于 DNA 甲基化分析，将 20 个 CAPA-IVM 囊胚与 12 个 COS 囊胚进行比较。对于 RNA-Seq 分析，将另一个单独的 15 个 CAPA-IVM 囊胚与 5 个 COS 囊胚进行比较。
参与者/材料、设置、方法：COS 胚胎来自 12 名符合 Rotterdam 标准的 PCOS 患者（根据 Rotterdam 标准），他们接受了常规卵巢刺激。对于 CAPA-IVM，23 名妇女接受了为期 3-5 天的高纯度 hMG（HP-hMG）治疗，在取卵前不给予 hCG 触发。卵母细胞首先在预成熟培养基（包含 C 型利钠肽的 CAPA 培养 24 小时）中培养，然后在含有 FSH 和 Amphiregulin 的 IVM 步骤（30 小时）中培养。在 ICSI 后，两组的第 5 天或第 6 天胚胎均进行玻璃化并用于 PBAT DNA 甲基化分析或单个胚胎的 RNA-seq 基因表达分析。从 PABT 和 RNA-seq 数据集中提取特定基因和 gDMR 的数据。
与 COS 胚胎相比，CAPA-IVM 囊胚在 gDMR 处显示出相似的甲基化和基因表达率。此外，两组的主要表观遗传调节剂的表达也相似。
局限性、谨慎的原因：COS 组的胚胎是在一系列培养基中产生的。CAPA-IVM 胚胎均使用相同的精子供体产生。纯体内来源的人类囊胚的 gDMR 中 DNA 甲基化水平尚不清楚。
CAPA-IVM 之后出生的儿童的随访很重要，因为它与其他新的 ART 一样，通常在没有对人类囊胚进行先前的表观遗传安全性研究的情况下引入临床实践。CAPA-IVM 为 PCOS 患者友好型 ART 开辟了新的前景。
研究资金/利益冲突：布鲁塞尔自由大学的 IVM 研究得到了以下机构的资助：比利时瓦隆大区创新署（Agentschap voor Innovatie door Wetenschap en Technologie-IWT，项目 110680）、弗兰德斯研究基金会（Fonds voor Wetenschappelijk Onderzoek-Vlaanderen-FWO-AL 679 项目，项目 G.0343.13）、比利时癌症基金会（HOPE 项目，Dossier C69Ref Nr 2016-119）和布鲁塞尔自由大学（IOF 项目 4R-ART Nr 2042）。G.K. 的实验室工作得到了英国生物技术和生物科学研究委员会和医学研究委员会的支持。作者没有利益冲突。
