Ovarian stimulation and egg retrieval: A control ovarian hyperstimulation (COH) protocol with human recombinant FSH (Gonal-F, Merck Serono S.A., Madrid, Spain) and hMG-HP (Menopur, Ferring S.A., Madrid, Spain) was initiated on menstrual cycle day 2 or 3 at a variable dose depending of age, body mass index (BMI) and antral follicular count (AFC). Pituitary suppression was achieved by daily s.c. administration of 0.25 mg of cetrorelix acetate (Cetrotide, Merck Serono, S.A., Madrid, Spain) when at least one follicle exceeded a mean diameter of 14 mm or estradiol was >400pg/mL. Ovulation was triggered by s.c administration of 6500U of hCG (Ovitrelle®, Merk-Serono, S.A., Madrid, Spain). Transvaginal oocyte retrieval was performed 36 hours after hMG trigger administration and ICSI was performed in all cases. Embryo quality was recorded according to ASEBIR criteria [27]. Luteal phase was supported by 400 mg/day of vaginal micronized progesterone (Progeffik, Lab. Effik, Madrid, Spain) starting at the day of embryo transfer. Pregnancy was defined as a positive pregnancy test; clinical pregnancy was defined as intrauterine sac visualization in transvaginal ecography. Collection of follicular fluid and granulosa cells: Follicular fluid (FF) were collected individually, after independent follicular puncture. FF and cumulus oophorus-oocyte complexes (COC) were subsequently separated after oocyte retrieval. Follicular fluid-derived cells were separated from erythrocytes by density gradient centrifugation on equal v/v of Histopaque (Sigma-Aldrich, St. Louis, USA) for 30min at 600g. Following centrifugation, three layers could be distinguished: a top layer containing the follicular fluid, a bottom layer containing erythrocytes and, in the middle, a ring-like layer containing the cells-derived from the follicular fluid. This middle layer was collected, washed by centrifugation for 10 min at 600g and resuspended in 1-ml phosphate-buffered saline (PBS). We use the cell strainer methodology to separate granulose cells, based on the fact that GCs form large cell aggregates in such a way that clusters can be retained by 40-µm pore cell strainers while erythrocytes and other blood contaminants (such as single cells and aggregates of a smaller size) pass through the strainer. Following the cell strainer methodology but with some modifications [PMID 10831550; 19001513; 22454458] the aspirates were first filtered through a 40-µm cell strainer (BD Biosciences) and the clusters of GCs were retained. The strainer was rinsed with 12-ml PBS in order to remove the last traces of blood contaminants and the strainer was then back washed with PBS to collect the ‘hypothetical’ GCs. The acquired suspension was incubated for several minutes and repeatedly aspirated through Pasteur pipettes stretched to different diameters to break up aggregates mechanically. The suspension was then filtered through a 70-µm cell strainer (BD Biosciences) to remove unwanted undispersed material, which was retained in the filter. The cell suspension, collected after filtering the sample, was subsequently washed by centrifugation for 5min at 600g, after which the supernatant was removed and the pellet was resuspended in 1 ml PBS.
Extracted molecule
total RNA
Extraction protocol
To ensure that miRNA fraction will be recovered we performed RNA extraction using the miRNeasy Kit (Qiagen, Valencia, CA, USA). RNA extracted was quantified using a NanoDrop (Thermo Fisher Scientific Inc, MA, USA) spectrophotometer and the proportion of miRNAs was evaluated using the Small RNA LabChip BioAnalyzer 2100, (Agilent Technologies Inc, DE, USA)
Label
Cy3-pCp
Label protocol
100ng of total RNA were mixed with spike and treated with phosphatase for 30' at 37º then cooled at 4C. Next DMSO was added and heated at 100ºC for 7.5' then cooled at 4ºC. Next mixed with Cy3-pCp and T4 RNA Ligase for 2 hours at 16C and cooled at 4ºC. Samples were dried in a vacuum for 3h at 45C. Then H2O added.
Hybridization protocol
After H2O resuspended, samples were mixed with another Spike, mixed with blocking agent and Hi-RPM hybridization buffer and heated at 100C for 5' and cooled at 4ºC. Then samples were loaded onto gaskets slides and microarrays slides were put on them. Finally Samples were hybridized for 20h at 55C.
Scan protocol
Microarrays slides were washed and scanned in Genepix Personal 4100A Scanner and scanned in Genepix Pro Software with a spot resolution of 5um in the green channel
Description
each sample was previous tested for miRNAs using Small RNA Labchip in an agilent bioanalyzer GV3
Data processing
Median intensity value of each spot data was normalized and log2 transformed using R software and libraries from bioconductor database. The intensity values were logarithmically and quartile normalized using R software and the Bioconductor libraries ‘limma’ and ‘tkrplot’. Next, replicated probes were further merged by mean using GEPAS.
