Correlation between oocyte cumulus cells gene expression, embryo morphokinetic, blastocyst formation and euploidy

Study question: Is cumulus cells (CC) gene expression pattern of five candidate genes, combined with morphokinetic parameters a non-invasive method to predict blastocyst formation and euploidy? Summary answer: CCs gene expression is correlated to embryo development and morphokinetic behaviour. Among blastocysts, we found PTGS2 over-expressed in CCs of euploid embryos. What is known already: Due to the crosstalk within the follicle, CC gene expression has been proposed as a non-invasive tool to predict oocyte/embryo competence. New promises come also from time-lapse, however no clear agreements on which gene and/or morphokinetic parameter can be used to predict embryo quality. A critical aspect of embryo quality is the blastocyst chromosomal status, and this feature can be used as an outcome measure to test potential biomarkers of oocyte quality. Study design, size, duration: Prospective longitudinal cohort study (September 2014–September 2015). A total of 58 CCs samples were individually collected from 21 consenting patients. Oocytes and belonging embryos where cultured individually in a time-lapse incubator (Embryoscope), until blastocyst stage. 29 oocytes (from 16 patients) reached blastocyst stage and underwent comprehensive chromosomal screening. Collected CCs samples were analysed for the expression of 5 candidate genes, previously found as predictive of oocyte competence: HAS2, PTGS2, CAMK1D, EFNB2, and STC1. Participants/materials, setting, methods: Advanced maternal age patients (38.3 2.5 years), underwent ICSI and pre-implantation genetic testing for aneuploidy. Severe male factor was excluded. All ovarian stimulations were performed with a fixed protocol. Retrieved cumulus-oocyte complexes were denuded individually. CCs gene expression was performed through qPCR using Taqman gene expression assays (Applied Biosystems). Twelve morphokinetic parameters were recorded during embryo culture. T-tests and Spearman’s rank correlation were performed through SigmaPlot (SSI, CA, USA). Main results and the role of chance: The 5 selected genes are known to be involved in cumulus expansion [hyaluronan acid synthase 2 (HAS2) and prostaglandin- endoperoxide synthase 2 (PTGS2)], in the mediation of developmental events [EphrinB2 (EFNB2) Ca-/calmodulin–dependent protein kinase 1 (CAMK1D)] and in autocrine and paracrine functions [Stanniocalcin-1 (STC1)]. Analysing CC gene expression of blastocysts versus arrested embryos, blastocyst formation was negatively correlated with EFNB2 expression (p 0.035; 95% CI: 0.0553–1.463. Logistic regression model adjusted for female age confirmed this association, OR 2.7, 95% CI 1.1–7.3. Combined analysis of morphokinetics and gene expression showed a negative correlation between timings of singamy and of second cell cycle and STC1 expression (p 0.004) while timings of 4-cell divisions were related to HAS2 expression (p 0.032). EFNB2, PTGS2 and CAMK1D expressions showed a positive correlation to timings of starting blastulation (p 0.015). Among obtained blastocysts we analyzed CC gene expression patterns of 18 aneuploid versus 11 euploid blastocysts, finding PTGS2 over-expression (p 0.026; 95% CI: 0.180–2.515) in euploid blastocysts. Limitations, reasons for caution: This study is limited to a specific population of patients, a relatively low number of CCs samples and a restricted number of genes. Results must be confirmed on larger scale Wider implications of the findings: We found a correlation between CC gene expression of 5 selected genes, morphokinetics and blastocyst formation while euploidy could only be related to PTGS2 expression. We confirmed the potentiality of CC gene expression analysis in predicting embryo competence. Trial registration number: None.