Investigation of Cumulus Cell Factors Associated With Embryo Development and Pregnancy Outcome in Human IVF

Recent evidence suggests that the expression of some candidate genes in cumulus cells (CC) have the potential to serve as markers of oocyte quality. The aims of this study were: 1) to validate a multiplex quantitative polymerase chain reaction (QPCR) method to measure four genes simultaneously in extracts from individual CC and; 2) to investigate the relationships in individual cumulus-oocyte-complexes (COC) amongst the expression levels of a range of candidate genes (N=8) from individual CC, the numbers of CC per COC and developmental indicators (good blastocyst development and live birth outcome) of the associated oocytes following in vitro fertilization (IVF) with intra-cytoplasmic sperm injection (ICSI). Sixty eight women were recruited for this study following approval from NZ Multi-Regional Ethics Committee and classified into four research groups: young and healthy women (<38 years, N=25), young women with polycystic ovarian syndrome (<38 years, N=11), young with diminished ovarian reserve (<38 years, N=12) and older and healthy women (≥40 years, N=20). Following exogenous rFSH-assisted ovarian stimulation, 608 COC were collected and subjected to ICSI. Oocyte and embryo quality, and pregnancy outcomes were recorded. Expression levels of the following candidate genes HAS2, FSHR, SLC2A4 (GLUT4), ALCAM, SFRP2, VCAN, NRP1 and PR in CC extracts from individual COC were measured by TaqMan QPCR and normalized against the house-keeping gene, RPL19. The numbers of CC from individual COC were calculated from RPL19 expression levels plotted against a standard curve of CC number. These results were then assessed against the outcomes of the associated oocytes following ICSI. HAS2, FSHR, ALCAM, VCAN, NRP1 and PR mRNA were detectable in most samples (98.5%) whereas those for SLC2A4 and SFRP2 were generally undetectable. The minimum number of CC required for QPCR was estimated to be ~70. The mean levels of FSHR mRNA were up-regulated in young women with PCOS compared to those in the other two groups of women <38y. Expression levels of HAS2 across all four groups of women were correlated to both biological (age, basal serum FSH and serum AMH) and treatment (amount of rFSH used for stimulation, follicle numbers and COC retrieved) variables. Investigations related to oocyte development in young and healthy women showed that 1) mean mRNA levels of VCAN, HAS2 and PR were higher (P=0.002, P<0.05, P<0.05, respectively) in CC associated with oocytes that resulted in good quality blastocysts and those of VCAN were higher (P