Collection and culture of first trimester decidual cells
Decidua was collected after informed consent at the time of elective termination of pregnancy or at the time of surgery for missed abortion. The Yale University School of Medicine Human Investigations Committee approved this protocol. Decidua was obtained electively from 23 individuals in the first trimester, 10 at the time of surgical intervention for spontaneous pregnancy loss and 13 undergoing elective first trimester termination. Tissue samples were divided in three for use in cell culture, real-time PCR or western analysis. For use in cell culture first trimester decidual stromal cells from these samples were isolated and purified to homogeneity on a Percoll gradient and passaged until >99% free of CD45+ cells as determined by fluorescent activated cell sorting analysis as previously described (Koopman et al., 2003
). The presence of decidual cells was confirmed as previously described and by detection of prolactin expression (Koopman et al., 2003
). Cells were grown to confluence in serum containing media. At confluence, the cells were primed with either 10−8
M 17β estradiol (E2
) or 10−8
M medroxyprogesterone acetate (MPA) for 7 days to simulate the steroidal milieu of pregnancy, and then cultured in serum-free defined medium containing the corresponding steroids with either 2.5 U/ml of thrombin or 1 ng/ml of IL-1β. After 24 h, total RNA was isolated (RNeasy Mini Kit, Qiagen, MD, USA).
RNA isolation and microarray analysis
Microarray analysis was used to assess the expression of all HOX genes in response to treatment. First trimester decidual cells from three patients were grown to confluence in Falcon T-25 flasks. The human Affymetrix array was used in triplicate for each experimental condition. Each in vitro experimental condition was repeated three times and in triplicate; each repetition used cells obtained from separate individuals. Cells were harvested with QIAzol™ lysis reagent (Qiagen, Valencia, CA, USA) and used to prepare total RNA. According to the manufacturer's instruction, 100 µg of total RNA was precipitated using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and used as a template for cDNA synthesis. A T7-(dT)24 oligo-primer was used to synthesize double-stranded cDNA by the One-Cycle cDNA Synthesis Kit (Affymetrix Inc., Santa Clara, CA, USA) which was subsequently purified using Sample Cleanup Module (Affymetrix Inc., Santa Clara, CA, USA) and ethanol precipitation. Then GeneChip IVT Labeling Kit (Affymetrix Inc., Santa Clara, CA, USA) was used to generate biotinylated cRNA. Additional cRNA purification was carried out using Sample Cleanup Module prior to the fragmentation of biotinylated cRNAs with 5× fragmentation buffer (Tris 200 mM, pH 8.1, KOAc 500 mM, MgOAc 150 mM). The chemically fragmented cRNAs were then hybridized on Affymetrix HG_U133 Plus 2.0 human chips in GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA, USA), screening for approximately 47 400 human genes and expression sequence tags, followed by fluorescence labeling with Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA) and optical scanning with Affymetrix GeneChip Scanner 3000 by W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University.
Raw data without normalization generated from Affymetrix GeneChip Operating Software Version 1.1.1 (GCOS 1.1.2) (Affymetrix, Santa Clara, CA, USA) were analyzed by GeneSpring software 7.2 (Agilent Technologies, Palo Alto, CA, USA). The gene readouts were normalized to the fiftieth percentile of the distribution of all measurements in each chip. Per gene normalization was performed using the median value of each gene throughout different chips in the same experimental condition. Nonparametric testing assuming unequal variance was applied to test statistical significance. Fold ratios were derived from comparing normalized data between control and treatment groups. HOX genes up- or down-regulated more than 2.0-fold by E2+MPA versus E2+MPA+IL-1β or thrombin were filtered.
Real-time polymerase chain reaction
HOX genes identified as expressed and regulated by IL-1β or thrombin in first trimester decidual cells by microarray analysis were confirmed using real-time RT–PCR. HOX gene expression after IL-1β or thrombin exposure were evaluated in cultured first trimester decidual cells. HOX gene expression in women undergoing surgical procedures was evaluated in surgical specimens of decidual tissue.
mRNA levels were evaluated by quantitative real-time RT–PCR and normalized to β-actin using the Roche LightCycler as previously described (Rackow and Taylor, 2009
). Briefly, the reactions were carried out using the LightCycler RNA Master SYBR Green I kit. Reaction conditions included 1.0 µg of RNA, 2 mM Mn[OAc]2
, respectively, 150 nM of each primer, and 1× RNA Master SYBR Green, for a final reaction volume of 20 µl. Primer sequences for each gene are as follows:
- HOXA1 5′-AGTTGGAGAGTACGGCTACCTG-3′ and 5′-TGCAGGGATGCAGCGATCTCCAC-3′
- HOXA3 5′-GGCTATCTGAACTCTATGCATTCG-3′ and 5′-AGGCCATGAGCGTGCGGGTCATA-3′
- HOXA9 5′-CTGTTCAACATGTACCTCACCA-3′ and 5′-CACTCGTCTTTTGCTCGGTC-3′
- HOXA10, 5′-AGGTGGACGCTGCGGCTAATCTCTA-3′ and 5′-GCCCCTTCCGAGAGCAGCAAAG-3′(46);
- HOXA11 5′-GTACTTACTACGTCTCGGGTCCAG-3′ and 5′-AGTCTCTGTGCACGAGCTCCT-3′
- β-actin, 5′-CGTACCACTGGCATCGTGAT-3′ and 5′-GTGTTGGCGTACAGGTCTTTG-3′.
Reverse transcription was carried out for 30 min at 61°C, followed by initial denaturation at 95°C for 30 s and 45 cycles including denaturation at 95°C for 2 s, annealing at 65°C (HOXA3, 9, 10 and 11), 62°C (β-actin), or 58°C (HOXA1) for 5 s and elongation at 72°C for 14 s. A melting curve was created following the amplification to observe the specificity of the primers. Each experiment was repeated in triplicate and performed three times using cells from 10 different individuals. Comparisons between in vitro
treatment groups were analyzed by ANOVA on ranks. Comparison of decidual HOX gene expression between 10 women experiencing spontaneous loss and 10 undergoing elective termination were performed using student's T
Western analysis was performed on 10 decidual samples obtained from women undergoing surgery for spontaneous pregnancy loss and 10 with a viable pregnancy. Protein was extracted from intact decidual tissue using Nuclear Extract Kit (Activemotif, CA, USA) according to manufacturer's instructions. Equal amounts of protein (60 µg) were electrophoresed through 4–15% polyacrylamide gels (Bio-Rad, CA, USA) at 160 V for 70 min and transferred onto Immun-Blot polyvindylidene difluoride membranes (Bio-Rad, CA, USA) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 100 V for 1 h. After incubation in blocking buffer (1× PBS, 0.2% Tween 20, 5% milk), the membrane was incubated individually with either goat polyclonal HOXA10 antibody (sc-17159) (Santa Cruz, CA, USA) dilution 1:200, or goat polyclonal Actin antibody (sc-1615) (Santa Cruz, CA, USA) dilution 1:1000 overnight at 4°C. After washing, the membranes were incubated for 1 h with biotinylated horse anti-goat secondary antibody (Vector, CA, USA) diluted (3.5 µg/ml) in the blocking buffer. The membranes were incubated in ABC Elite (Vector, CA, USA), and then stained by DAB (Vector, CA, USA). Negative controls demonstrated lack of reactivity in tissues that do not express HOXA10 or HOXA11.