DNA methylation and mRNA expression of imprinted genes in blastocysts derived from an improved in vitro maturation method for oocytes from small antral follicles in polycystic ovary syndrome patients.

STUDY QUESTION: 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? SUMMARY ANSWER: No significant differences in imprinted DNA methylation and gene expression were detected between COS and CAPA-IVM blastocysts. WHAT IS KNOWN ALREADY: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. WIDER IMPLICATIONS OF THE
FINDINGS: 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.