Human antral follicles <6 mm: a comparison between in vivo maturation and in vitro maturation in non-hCG primed cycles using cumulus cell gene expression

abstract: Within the context of an oocyte in vitro maturation (IVM) program for reproductive treatment, oocyte cumulus complexes (COCs) derived from follicles ,6 mm in patients with PCOS were matured in vitro. Key transcripts related to meiotic maturation (FSHR, LHCGR, EGFR, PGR) and oocyte competence (AREG, ADAMTS, HAS2, PTGS2) were quantified in cumulus cells (CCs) before and after maturation. Control CC samples were collected from PCOS and normo-ovulatory patients who had undergone conventional gonadotrophin stimulation for IVF/ICSI. Additional control samples from a non-stimulated condition were obtained ex vivo from patients undergoing ovariectomy for fertility preservation. Expression data from CCs from follicles with a diameter of ,6 mm before (IVM-CCs) and after in vitro maturation (IVM-CCs) were obtained after pooling CCs into four groups in relation to the percentage of matured (MII) oocytes obtained after 40 h of IVM (0; 40 -60; 61-80; 100% MII) and values were compared with in vivo matured controls (IVO-CCs). Genes encoding key receptors mediating meiotic resumption are expressed in human antral follicles of ,6 mm before and after IVM. The expression levels of FSHR, EGFR and PGR in CCs were significantly down-regulated in the IVO-CCs groups and in the 100% MII IVM group compared with the BM groups; all the receptors studied in the 100% MII IVM group reached an expression profile similar to that of IVO-CCs. However, after maturation in a conventional IVF/ICSI cycle, IVO-CCs from large follicles contained significantly increased levels of ADAMTS1, AREG, HAS2 and PTGS2 compared with IVM-CCs and IVM-CCs; the expression patterns for these genes in all IVMCCs were unchanged compared with IVM-CCs. In conclusion, genes encoding receptors involved in oocyte meiotic resumption appeared to be expressed in CCs of small human antral follicles. Expression levels of genes-encoding factors reflecting oocyte competence were significantly altered in IVM-CCs compared with in vivo matured oocytes from large follicles. Observed differences might be explained by the different stimulation protocols, doses of gonadotrophin or by the intrinsic differences between in vivo and in vitro maturation

Chromosome constitution of human embryos generated after in vitro maturation including 3-isobutyl-1-methylxanthine in the oocyte collection medium

STUDY QUESTION: Do cleavage-stage embryos obtained from oocytes matured in vitro after pre-incubation with a phosphodiesterase inhibitor (IBMX) carry more chromosomal abnormalities than those generated from oocytes matured in vivo? SUMMARY ANSWER: The rate and type of chromosomal abnormalities in normally developing cleavage-stage embryos generated with an in vitro maturation (IVM) system including pre-incubation with IBMX are not different from those observed in supernumerary embryos obtained from oocytes matured in vivo. WHAT IS KNOWN ALREADY: Very limited information is available about the chromosomal constitution of IVM embryos. Previous studies were carried out using FISH on single biopsied blastomeres or arrested whole embryos and only provided fragmentary information on chromosomal abnormalities in IVM embryos. There is no systematic study of chromosomal abnormalities in all blastomeres of human Day 3 embryos with good morphology. STUDY DESIGN, SIZE, DURATION: Between July 2012 and December 2012, 16 young (age <35 years old) egg donors underwent 18 IVM cycles for the generation of research embryos. Eighteen embryos developed to Day 3 and were analysed using array comparative genomic hybridization (aCGH). PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature oocytes were retrieved from 2 to 10 mm follicles after mild ovarian stimulation with gonadotrophins but without hCG ovulation trigger. At collection, oocytes were pre-incubated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and matured in vitro. After IVM culture, mature oocytes were microinjected with sperm from a single donor. Embryos were cultured to Day 3 after ICSI and all blastomeres of 18 good-morphology embryos were collected individually for aCGH. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte maturation rate in vitro was 50.2% (120/239). The mean fertilization rate was 68.3% (82/120) and 30.5% (25/82) of fertilized oocytes developed into a morphologically good quality embryo on Day 3 after ICSI. Of these, 18 embryos that developed well up to Day 3 were analysed using aCGH. Eighty of the 123 blastomeres analysed showed at least one chromosomal abnormality. Three out of eighteen embryos had completely normal cells. A single embryo carried a meiotic abnormality, 11 embryos were mosaic and three were chaotic. Although the aneuploidy data of this study are too limited to allow statistical analysis, these data are comparable to our own published data on the chromosome constitution of whole day 3 and day 4 embryos after conventional ART. LIMITATIONS, REASONS FOR CAUTION: Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. Although the limited number of embryos analysed here precludes firm conclusions, they provide valuable data on possible causes of the reduced potential of IVM embryos. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to describe the complete chromosome complement of all single blastomeres of good-morphology day 3 embryos obtained with IVM (including the presence of IBMX in a pre-incubation medium). The results demonstrate that a high proportion of good-morphology embryos are aneuploid and that there is no obvious increase in aneuploidies as a result of IVM which seems to suggest that the reduced efficiency of IVM technology compared with standard IVF may be accounted for by factors other than aneuploidy, such as cytoplasmic defects or reduced endometrial receptivity. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the TBM (Applied Biomedical Research with Societal Finality) programme of the IWT (Agency for Innovation through Science and Technology - Flanders, 110680) and by a Methusalem grant of the Vrije Universiteit Brussel. C.S. is a post-doctoral fellow of the Fund for Scientific Research Flanders (FWO - Vlaanderen). K.J. is a PhD student funded by the FWO. The University of Adelaide owns a patent family associated with IVM technologies that is licensed to Cook Medical. R.B.G. and J.G.T. are inventors. The remaining authors have no conflict of interest to declare.C. Spits, L. Guzman, A. Mertzanidou, K. Jacobs, C. Ortega-Hrepich, R.B. Gilchrist, J.G. Thompson, M. De Vos, J. Smitz, and K. Sermo