P-422: Granulosa cell gene expression of IGF-II and IGFBP-3 in PCOS patients as a predictor of IVF outcome

Abstract

IGF-II is the predominant intraovarian IGF and its synthesis is hormonally-sensitive (Proc Nat Acad Sci, 1987). IGFBP-3 is the primary intraovarian binding protein and is present in dominant follicles (JCEM 1993,1994). Our objective was to examine the association between granulosa cell gene expression of IGF-II and IGFBP-3 and oocyte maturity in a cohort of PCOS patients undergoing IVF. Analysis of granulosa cell mRNA from PCOS and male factor infertility (control) patients undergoing IVF/ICSI with OCP/GnRH agonist dual suppression followed by low dose stimulation (<2amps/day). Nine PCOS patients and 9 normally ovulating controls were recruited with IRB approval. Follicular fluid aspirates were obtained following removal of oocyte-cumulus complexes. Samples were centrifuged, and granulosa cells were prepared and isolated with 50% Percoll. For each patient, half of the cells were cultured for 5 days in uniform conditions (1:1 DMEM:F12 with 10% fetal calf serum and 0.1% Gentamicin)(“cultured”), and half of the cells underwent immediate RNA extraction (“fresh”). RNA extraction was performed with Trizol reagent. Following cDNA synthesis, real-time quantitative PCR was performed with IGF-II and IGFBP-3 primers and probes (Applied Biosystems). Samples were run in duplicate. For PCR we report Ct values which decrease linearly with increasing target quantity and reflect input sample concentration. PCOS patients had a higher mean BMI, but stimulation parameters and outcome were comparable (Table 1). Mean expression of IGF-II and IGFBP-3 was comparable between PCOS and control patients (Figure 1). Adjustment for BMI did not affect this finding. When individual patients’ cultured and fresh cells’ gene expression was compared, cultured cells demonstrated significantly increased expression. Ct levels of IGF-II for freshly cultured cells correlated negatively with peak E2 levels in controls (R= -0.99, p=0.05) and PCOS (R= -0.84, p=0.07), suggesting that higher granulosa cell IGF-II production correlated with higher peak E2 levels. IGF-II and IGFBP-3 expression did not correlate with number of oocytes retrieved, oocyte maturity, or ampules of gonadotropins. Gene expression did not differ between pregnant and nonpregnant patients.Tabled 1View Large Image Figure ViewerDownload (PPT) Although no significant differences were found between PCOS and controls’ expression of IGF-I and IGFBP-3, real-time quantitative PCR offers the potential to detect differences between PCOS and control granulosa cells both in vivo and in vitro. Study of a greater number of patients will hopefully allow further insight into the PCOS granulosa cell gene expression.