The MNIVF group of patients underwent transvaginal ultrasonographic examination on day 9 of the cycle. Measurement of serum oestradiol and testing of urine for LH surge presence was performed at the same time. For serum oestradiol measurement, a commercially available kit (Oestradiol-2; Sorin Biomedica, Saluggia, Italy) was used. The detection of LH surge was also made with a commercially available kit (RapiTest LH; Morwell Diagnostics GmbH, Egg, Switzerland). As soon as the dominant follicle reached ≥ 16 mm in diameter, serum oestradiol exceeded 0,39 nmol/l and there was no LH surge detected in urine sample, 5000 IU of human chorionic gonadotrophin (HCG) (Pregnyl; N.V. Organon, Oss, The Netherlands) was injected subcutaneously. Ultrasound-guided transvaginal oocyte aspiration was performed 31-32 h after HCG administration. The GnRH agonists group of patients buserelin acetate (Suprefact; Hoechst AG, Frankfurt/Main, Germany) was administered subcutaneously from day 22, with a daily dose of 0.6ml (600pg). When criteria for ovarian desensitization were fulfilled (serum oestradiol <0.05nmol/l, follicles <5mm in diameter), patients were subcutaneously administered 225 IU of gonadotrophin folitropin alpha (Gonal F; Industria Farmaceutica Serono S.p.A., Bari, Italy). When at least three follicles were ≥17mm in diameter and serum oestradiol exceeded 0.40nmol/l per follicle, 10.000 IU of HCG (Pregnyl; N.V. Organon) was administered. Ultrasound guided transvaginal oocyte aspiration was performed 34-36h after HCG administration. In the GnRH antagonist group 225 IU of gonadotrophin folitropin alpha (Gonal F; Industria Farmaceutica Serono S.p.A., Bari, Italy) was administered subcutaneously on day 2 of the cycle. The GnRH antagonist cetrorelix acetate (Cetrotide; Asta Medica AG, Frankfurt, Germany) was administered subcutaneously at a dose of 0,25mg when the dominant follicle measured 13mm in diameter. When at least three follicles were ≥17mm in diameter and serum oestradiol exceeded 0.40nmol/l per follicle, 10.000 IU of HCG (Pregnyl; N.V. Organon) was administered. Ultrasound guided transvaginal oocyte aspiration was performed 34-36h after HCG administration. Immediately after cumulus-oocyte complex (COC) retrieval, a part of cumulus cells (CC) surrounding a single oocyte was removed by using a sharp needle. Individual CC samples were washed in PBS, snap-frozen in liquid nitrogen and stored in vials at -80°C until RNA isolation. The oocytes were cultured individually in Universal IVF medium (MediCult a/s, Jyllinge, Denmark) and fertilization was carried out 2 h after oocyte retrieval. After 24 h, fertilization status was assessed. Embryos were later cultured in a sequential media (BlastAssistSystem; MediCult, Denmark) for five days and one or two embryos at the stage of morulae or blastocyst were transferred on day 5; supernumerary good quality blastocysts were cryopreserved. Implantation was assessed 14 days after embryo transfer using serum β- HCG measurement. To confirm intrauterine pregnancy and the number of gestational sacs with heartbeat activity, transvaginal ultrasound was performed 4 weeks after embryo transfer.
Growth protocol
Not applicable.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRI reagent (Sigma– Aldrich, St. Louis, USA) according to manufacturer’s instruction with small modification due to the small sample volume. Glycogen (Ambion, Woodward, USA) was used as a carrier to increase RNA yield. Briefly, CC from individual follicle were homogenized in 500 μL TRI reagent supplemented with 125 μg of glycogen. After incubation at room temperature, 100 μL chloroform was added and the sample was vortexed vigorously. After 15 min centrifugation at 12,000 x g and 4°C, RNA was transferred from aqueous phase and precipitated with 250 μL of isopropanol. After 25 min centrifugation at 12,000 x g and 4°C, RNA pellet was washed 3 times with 75% ethanol. Total RNA was resuspended in 15 μL of RNase-free water and stored at -80°C. The integrity of RNA was assessed on Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) to assure high quality of total RNA. Total RNA quantity and purity was measured with a Nanodrop ND-1000 spectrophotometer (Nanodrop Techonologies Inc., Wilmington, DE, USA).
Label
biotin
Label protocol
200 ng of RNA was amplified and converted to cDNA using the WT Expression Kit (Ambion, Austin, USA). The resulting cDNA was fragmented and labeled using the GeneChip WT Terminal Labeling and Controls Kit (Affymetrix, Santa Clara, USA).
Hybridization protocol
GeneChip Human Gene 1.0 ST Arrays (Affymetrix, Santa Clara, USA) were hybridized for 16 hours and washed using GeneChip Fluidics Station 450 according to manufacturer’s recommendations.
Scan protocol
GeneChips were scanned using the Affymetrix Scanner GCS 3000 7G .
Description
Cumulus cells (CC) gene expression of mature, metaphase II (MII) oocytes that developed to the stage of blastocyst on day 5 of IVF procedure from GnRH Agonist treated patient AH.
Data processing
Data were normalized using RMA algorithm from R/Bioconductor/XPS package. HuGene-1_0-st-v1.r4.pgf HuGene-1_0-st-v1.na30.1.hg19.probeset.csv
