score: morula oxygen concentration: High embryoid: 11_3 medium: HTF age mother: 30
Treatment protocol
During the experimental culture, the two culture media were prepared according to the manufacturers’ instructions. HTF-medium was supplemented with 10% GPO and was equilibrated in 5% CO2, while G5-medium (Vitrolife, Göteborg, Sweden) was supplemented with HSA (5 mg/ml) and was equilibrated in 6% CO2. Randomization of the women to the four experimental groups was performed in the morning before thawing using sealed envelopes and stratification was performed for maternal age (≤ 35 and >35 years). The embryos were morphologically assessed according to the Gardner criteria, by two (SR, SM) embryologists that were blinded for the culture conditions used, right after thawing, at the end of the first day of culture and at the end of the second day of culture just before freezing and parameters like cell number, percentage of fragmentation and presence of dead cells were noted (ref Gardner, et al., 2000). At the end of the culture, individual embryos (n=89) were transferred in PCR-tubes, flash frozen in liquid nitrogen and stored at -80oC until RNA extraction. Only embryos that had developed to the morula and blastocyst stage (n=41) were further analysed.
Growth protocol
All embryos were cultured in HTF (Lonza, Verviers, Belgium) supplemented with 10% GPO (XX) under 5% CO2 and 20% O2 as part of normal patient treatment until day 4 when they were cryopreserved. Forty women (23 undergoing IVF and 17 undergoing ICSI) were randomised to have their embryos cultured for two additional days after thawing (till embryonic day 6) in one of the following groups: (i) HTF-medium and 20% O2, (ii) HTF-medium and 5% O2, (iii) G5-medium and 20% O2 and (iv) G5-medium and 5% O2.
Extracted molecule
total RNA
Extraction protocol
For sample preparation we used a protocol that was recently developed and validated for analysis of picogram amounts of RNA (Mantikou, et al. 2013, PLOS). Briefly, RNA isolation was performed using the Arcturus PicoPure isolation kit (Applied biosystems) following the instructions of the manufacturer. RNA extraction was performed with 50μl extraction buffer and the recommended DNase treatment was performed. The RNA was then amplified using the Arcturus RiboAmp HS Plus (Applied Biosystems). The concentration of the aRNA was measured on the Nano-drop ND-1000 (Thermo Scientific) and the quality of the amplified product was investigated with the BioAnalyzer (Agilent Technologies) using the RNA Nano 6000 kit (Agilent Technologies). 5 µg of amplified RNA was dried in a speedvac and then dissolved in 5 µl 50 mM carbonate buffer (pH 8.5) and placed at 370C for 10 min.
Label
Cy3
Label protocol
10 µl of CyDye (Cy3 for test samples and Cy5 for reference samples) diluted in DMSO was added to each reaction. Then, the samples were incubated in the dark at room temperature for 60 min. The reactions were quenched by 5 µl of 4 M hydroxylamine for 15 min at room temperature in the dark. The labeled RNA was purified according to the MicroElute RNA Cleanup protocol (Omega). The RNA was finally eluted in 15 µl Rnase/DNase free water. Pooled aRNA from the amplified samples was used as a reference in all experiments. The yield and CyDye incorporation of labeled aRNA were measured with the NanoDrop ND-1000 (Thermo Scientific).
During the experimental culture, the two culture media were prepared according to the manufacturers’ instructions. HTF-medium was supplemented with 10% GPO and was equilibrated in 5% CO2, while G5-medium (Vitrolife, Göteborg, Sweden) was supplemented with HSA (5 mg/ml) and was equilibrated in 6% CO2. Randomization of the women to the four experimental groups was performed in the morning before thawing using sealed envelopes and stratification was performed for maternal age (≤ 35 and >35 years). The embryos were morphologically assessed according to the Gardner criteria, by two (SR, SM) embryologists that were blinded for the culture conditions used, right after thawing, at the end of the first day of culture and at the end of the second day of culture just before freezing and parameters like cell number, percentage of fragmentation and presence of dead cells were noted (ref Gardner, et al., 2000). At the end of the culture, individual embryos (n=89) were transferred in PCR-tubes, flash frozen in liquid nitrogen and stored at -80oC until RNA extraction. Only embryos that had developed to the morula and blastocyst stage (n=41) were further analysed.
Growth protocol
All embryos were cultured in HTF (Lonza, Verviers, Belgium) supplemented with 10% GPO (XX) under 5% CO2 and 20% O2 as part of normal patient treatment until day 4 when they were cryopreserved. Forty women (23 undergoing IVF and 17 undergoing ICSI) were randomised to have their embryos cultured for two additional days after thawing (till embryonic day 6) in one of the following groups: (i) HTF-medium and 20% O2, (ii) HTF-medium and 5% O2, (iii) G5-medium and 20% O2 and (iv) G5-medium and 5% O2.
Extracted molecule
total RNA
Extraction protocol
For sample preparation we used a protocol that was recently developed and validated for analysis of picogram amounts of RNA (Mantikou, et al. 2013, PLOS). Briefly, RNA isolation was performed using the Arcturus PicoPure isolation kit (Applied biosystems) following the instructions of the manufacturer. RNA extraction was performed with 50μl extraction buffer and the recommended DNase treatment was performed. The RNA was then amplified using the Arcturus RiboAmp HS Plus (Applied Biosystems). The concentration of the aRNA was measured on the Nano-drop ND-1000 (Thermo Scientific) and the quality of the amplified product was investigated with the BioAnalyzer (Agilent Technologies) using the RNA Nano 6000 kit (Agilent Technologies). 5 µg of amplified RNA was dried in a speedvac and then dissolved in 5 µl 50 mM carbonate buffer (pH 8.5) and placed at 370C for 10 min.
Label
Cy5
Label protocol
10 µl of CyDye (Cy3 for test samples and Cy5 for reference samples) diluted in DMSO was added to each reaction. Then, the samples were incubated in the dark at room temperature for 60 min. The reactions were quenched by 5 µl of 4 M hydroxylamine for 15 min at room temperature in the dark. The labeled RNA was purified according to the MicroElute RNA Cleanup protocol (Omega). The RNA was finally eluted in 15 µl Rnase/DNase free water. Pooled aRNA from the amplified samples was used as a reference in all experiments. The yield and CyDye incorporation of labeled aRNA were measured with the NanoDrop ND-1000 (Thermo Scientific).
Hybridization protocol
825 ng of labeled sample aRNA and 825 ng labeled reference aRNA were hybridized to 4x 180k custom designed Agilent arrays (Agilent technologies). The 180k microarray (design ID:028004) contained ~42.000 probes in quadruplet and 11.200 control probes (ref Mantikou, et al., 2013, PLOS). The hybridization mixture was prepared according to the Two-Color Microarray-Based Gene Expression Analysis protocol (Agilent technologies). The aRNA was allowed to hybridize for 17 hours at 650C and 10 rpm.
Scan protocol
Afterwards, the slides were washed and scanned in an ozone-free room with the Agilent scanner GA2505C (Agilent Technologies). Slides were scanned at 3 μm resolution.
Data processing
Microarray data were extracted with Feature Extraction software 10.7.1 (Agilent Technologies). The data were analyzed in R-2.14.1 (http://cran.r-project.org/). All arrays were subjected to a set of quality control checks, such as visual inspection of the scans, checking for spatial effects through pseudo-color plots, and inspection of pre- and post-normalized data with box plots, density plots, ratio-intensity plots and principal component analysis First the data were normalized using lowess normalization using both the test and reference channels then expression values were calculated by using the robust multi-array average (RMA) algorithm (Irizarry et al.). When multiple embryos per woman were correlated only one of those samples was selected to avoid effects of pseudoreplication. For each experimental condition mean expression values were calculated. The conditions were compared using Spearman correlation coefficients.
