Pubblicazioni Follicle-specific predisposition to aneuploidy as revealed by transcriptomic analysis of cumulus cells

INTRODUCTION: Oogenesis consists of two meiotic divisions, and two different stages during which the maturing oocytes arrests. In the case of the first meiotic division this arrest can last for 4-5 decades. Therefore a strict regulatory mechanism is necessary to ensure that both cytoplasmic and nuclear events of oocyte maturation occur in an orderly and coordinated manner. Cumulus cells (CCs) are differentiated granulosa cells which surround the maturing oocyte in antral follicles and play a crucial role in regulating oocyte maturation. This takes place via the establishment of a bi-directional communication between the oocyte and its surrounding CCs with the formation of gap junctions. Female meiosis is prone to chromosome malsegregation errors leading to aneuploidy, which increases dramatically with advancing age. Although genetic analyses can reveal the presence of lethal chromosome anomalies in oocytes and embryos, the invasive nature of such procedures may have an adverse impact on embryonic viability and are not permitted in all countries due to ethical concerns. Our study aimed to: 1. Compare the gene expression patterns of CCs associated with normal and aneuploid oocytes; 2. Obtain an insight into the follicular environment of oocytes that become aneuploid; 3. Identify novel non-invasive markers of aneuploidy and general oocyte physiology and competence.

MATERIALS & METHODS: Oocyte chromosome status was determined by first polar body (PB) analysis using microarray comparative genomic hybridization (aCGH), identifying aneuploidy affecting any chromosome. CC gene expression was quantified via microarray and results for 94 candidate genes were verified using real-time polymerase chain reaction.

RESULTS: A total of 26 first PBs and CCs removed from their corresponding oocytes were examined during the course of our study. These were donated by nine women of an average age of 38.3 years (range: 32-46 years), who were undergoing IVF due to male factor infertility and tubal problems (i.e. no ovarian involvement). The expression of approximately 30,000 genes was examined via microarray analysis of CCs from normal and aneuploid oocytes. The observed expression patterns were very similar among all CCs, with 17,388 genes being consistently detected in all samples. There were however, statistically significant differences in the expression of 729 genes (P<0.05) seen between the CCs of normal and aneuploid groups. The vast majority of these genes (457) were downregulated in the CCs associated with aneuploid oocytes. Interestingly, 14 of these genes showed highly significant expression differences (P<0.01) and large mRNA transcript copy number fluctuations (>4).  The differential expression of most of these genes was verified via real-time PCR. Several genes involved in transcription and translation regulation were under-expressed in the CCs of the aneuploid oocytes. Moreover, various signalling pathways including some involved in cellular metabolism and apoptosis were also seen to be malfunctioning in the CCs of the aneuploid oocytes.

CONCLUSIONS: The comparison of the gene expression patterns seen in CCs surrounding normal and chromosomally abnormal oocytes provided for the first time an insight into the follicular environment of oocytes that become aneuploid. From the obtained results it was evident that the CCs of the aneuploid oocytes were less transcriptionally active and not as proliferative compared to the CCs of the normal oocytes. Additionally, genes involved in pathways related to hypoxia, hormonal response and apoptosis were abnormally expressed, suggesting a causal relationship between follicular microenvironment, specifically levels of steroid hormones and oxygenation, and aneuploidy. Among the 729 differentially expressed genes we identified 14 which consistently showed highly significant expression differences between the two groups of samples. These genes have the potential to serve as possible non-invasive markers of oocyte aneuploidy, either reducing or possibly eliminating the necessity for polar body and/or blastomere biopsy.

E. Fragouli, Z. Huang, V. Bianchi, A. Borini, U. Kayisli, P. Patrizio, D. Wells (2011), 27th Annual Meeting of the European Society for Human Reproduction and Embryology (ESHRE) Stockholm, 4-5-6 July, 2011.

in attesa di pubblicazione su Human Reproduction

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