Differential gene expression in human granulosa cells from recombinant FSH versus human menopausal gonadotropin ovarian stimulation protocols

Abstract

Background: The study was designed to test the hypothesis that granulosa cell (GC) gene expression response differs between recombinant FSH and human menopausal gonadotropin (hMG) stimulation regimens.

Methods: Females < 35 years-old undergoing IVF for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, GnRH agonist long protocol plus individualized dosages of (1) recombinant (r)FSH (Gonal-F) or (2) purified human menopausal gonadotropin (hMG; Menopur). Oocytes were retrieved 35 h post-hCG, and GC were collected. Total RNA was extracted from each GC sample, biotinylated cRNA was synthesized, and each sample was run on Human Genome Bioarrays (Applied Microarrays). Unnamed genes and genes with <2-fold difference in expression were excluded.

Results: After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, approximately 180 as transcriptional regulators, and approximately 175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11), bone morphogenetic protein receptor II (BMPR2), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-4, IGFBP-5, and hypoxia-inducible factor (HIF)-1 alpha.

Conclusions: Results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence.

Figure 1

Differentially expressed genes by functional category. Black bars represent genes with greater expression in rFSH-stimulated GC (n = 4); gray bars represent genes with greater expression in hMG-stimulated GC (n = 3).

Figure 2

Comparison of mean relative (hMG:rFSH) gene expression of selected genes by DNA microarray (gray bars) and RT-PCR (black bars). The expression of each gene of interest was calculated relative to that of GAPHD, then the expression in hMG-treated samples was divided by that of the rFSH-treated samples. For both sets of data, the values represent the ratios of the group means.