Cumulus cells gene expression profiling in terms of oocyte maturity in controlled ovarian hyperstimulation using GnRH agonist or GnRH antagonist

Rok Devjak, Klementina Fon Tacer, Peter Juvan, Irma Virant Klun, Damjana Rozman, Eda Vrtačnik Bokal, Rok Devjak, Klementina Fon Tacer, Peter Juvan, Irma Virant Klun, Damjana Rozman, Eda Vrtačnik Bokal

Abstract

In in vitro fertilization (IVF) cycles controlled ovarian hyperstimulation (COH) is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH) agonists or antagonists, to prevent premature luteinizing hormone (LH) surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC) in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI) and mature metaphase II (MII) oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2), follicle stimulating hormone receptor (FSHR), vascular endothelial growth factor C (VEGFC) and serine protease inhibitor E2 (SERPINE2). Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA). No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Experimental design.
Figure 1. Experimental design.
number of patients included in the study with respect to the two GnRH analogue treatments and the number of collected CC samples; CC MI: cumulus cells of metaphase I oocytes; CC MII-NF: cumulus cells of unfertilized metaphase II oocytes; CC MII-BL: cumulus cells of metaphase II oocytes developed to the blastocyst stage.
Figure 2. Schematic representation of microarray data…
Figure 2. Schematic representation of microarray data analysis according to oocyte maturity.
Boxes represent CC samples at different oocyte stages and their numbers; large box represents CC samples merged from MII-NF and MII-BL oocytes. Contrasts and corresponding numbers of differentially expressed genes are shown in circles. CC MI: cumulus cells of metaphase I oocytes; CC MII-NF: cumulus cells of unfertilized metaphase II oocytes; CC MII-BL: cumulus cells of metaphase II oocytes developed to the blastocyst stage; CC MII-NF vs. CC MI: contrast between CC MII-NF and CC MI; CC MII-BL vs. CC MI: contrast between CC MII-BL and CC MI; CC MII-BL vs. CC MII-NF: contrast between CC MII-BL and CC MII-NF; CC MII vs. CC MI: contrast between CC MII and CC MI.
Figure 3. Subgroup of genes connected to…
Figure 3. Subgroup of genes connected to follicle stimulating hormone (FSH) and luteinizing hormone (LH) from a top ranked gene network.
It is associated with Post-Translational Modification, Cellular Development, Cellular Growth and Proliferation as identified by Ingenuity Pathways Analysis. Top ranked gene network was obtained from comparisons of differential expression between CC MII and CC MI. Genes are represented as nodes, and the biological relationship between two nodes is represented as edge (line): a plain line indicates direct interaction, a dashed line indicates indirect interaction; a line without arrowhead indicates binding only, a line finishing with a vertical line indicates inhibition; a line with an arrowhead indicates ‘acts on’. The green colour intensity of the nodes indicates the degree of down-regulation in the CC MII group. CC MI: cumulus cells of metaphase I oocytes. CC MII: cumulus cells of metaphase II oocytes.
Figure 4. Expression (log2 fold change) of…
Figure 4. Expression (log2 fold change) of VEGFC, SERPINE2, AMHR2 and FSHR from microarrays and qPCR showing contrast between CC MII and CC MI.
Bars represent expression of CC MII vs. CC MI as log2 fold change with standard deviation. Blue represents microarray data and red qPCR data. Correlation coefficient (r = 0.98) and p value (p = 0.02) indicate strong and significant correlation between microarray and qPCR data. Corr r: Pearson correlation coefficient with p value between microarray and qPCR values. CC MI: cumulus cells of metaphase I oocytes. CC MII: cumulus cells of metaphase II oocytes.

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