OCT-4 expression in follicular and luteal phase endometrium: a pilot study

We performed a prospective, single center cohort study between September 2006 and March 2007 in a population of 89 consecutive patients undergoing hysteroscopy and endometrial sampling at the Endoscopy Unit of the Department of Gynecologic Endocrinology and Reproductive Medicine at Vienna Medical University, Vienna, Austria. The mean age of the patients was 33.9 ± 5.2 years. All women had regular menstrual cycles during the last 6 months (menstrual cycle length 25-35 days) and did not take any hormone therapy. Menstrual phase assessment was based on the date of the last menstrual period with the luteal and follicular phases determined by halfing the median number of cycle days of the last three menstrual cycles of the patient and confirmed by histopathological analysis of the endometrial specimen. Indications for surgery were primary sterility (n = 32), secondary sterility (n = 34), endometriosis and/or dysmenorrhea (n = 10), recurrent pregnancy loss (n = 4), chronic pelvic pain (n = 4), and others (n = 5). Written informed consent was obtained by all patients.

For RNA extraction frozen tissue samples were triturated and total RNA was extracted using the TRI REAGENT method (Molecular Research Centre, Inc., OH, USA). RNA concentration was determined by measuring the optical density at 260 nm. 1 μg RNA was reversely transcribed into first strand complementary DNA (cDNA) using Superscript (Invitrogen Ltd., Paisley, UK). The resulting cDNA was amplified by polymerase chain reaction (PCR) using primers specific for OCT-4 [11]. The following primers were used for RT-PCR reactions: OCT 4 forward 5'-GAC AAC AAT GAA AAT CTT CAG GAG A-3' and OCT reverse 5'-TTC TGG CGC CGG TTA CAG AAC CA-3'. The PCR was started with a denaturing step at 94°C for 5 min and the amplification of the 218 bp product was performed for 35 cycles at 94°C for 30 sec, 61°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 10 min. As control for genomic DNA we used extracted RNA only (no cDNA). As positive control for the expression of OCT -4 we used RNA from embryonic carcinoma of testis. Human glyceraldehyd-3-phosphate dehydrogenase (GAPDH) was amplified in parallel reactions as a housekeeping reference gene serving as an internal control for the quantity and quality of the cDNA. Primers for GAPDH were: forward 5'-TCT GGT AAA GTG GAT ATT GTT G-3' and reverse 5'-GAT GGT GAT GGG ATT TCC-3'. The amplified product for GAPDH has a size of 156 base pairs. Amplified samples were separated on 1.5% agarose gels in the presence of ethidium bromide and visualized by the Eagle Eye System (Stratagene, Amsterdam, The Netherlands). Quantitative analysis was performed by densitometric scanning of the gels using the AlphaDigiDoc 1000 software (Alpha Innotech Corp., San Leonardo, CA). OCT-4 mRNA levels were determined by calculating the OCT-4/GADPH ratios (Figure 1). We optimized all PCR steps with respect to sensitivity, reproducibility, and linearity: different template, enzyme, and primer concentrations, reaction times, and temperatures were tested. The resulting optimal conditions are given above. Since the technician and the scoring person were both blinded to the menstrual phase of the specific samples, the samples have been placed in the order of sampling and not arranged according to menstrual phase.

Although the mRNA quantities varied, expression of OCT-4 was detected in all samples analyzed (Figure 2). By including control reactions with input RNA instead of cDNA obtained after reverse transcription of RNA, we are confident that the OCT-4 PCR product of the endometrial samples is not due to genomic DNA contamination.

Increased mRNA levels were evaluated relative to GAPDH, ie mRNA levels equivalent or less than GAPDH were considered normal, mRNA levels above the levels measured for GAPDH were considered indicative of increased transcription.

For confirmation of OCT-4 expression in endometrial tissue, we performed Western Blot experiments, but were not able to consistently demonstrate OCT-4 protein in the frozen tissue samples used (see Additional File 1, Figure S1).

We used formalin-fixed, paraffin-embedded tissue samples of endometrium from 89 patients. From these sections we performed immunohistochemical staining using a primary antibody recognizing OCT-4 (OCT-4 [C-10]: sc-5279, Santa Cruz Biotechnology, California) as previously described [12]. Stained sections were scored according to the immunoreactive score (IRS) described by Remmele and Stegner [17]. The IRS results from the multiplication of a staining intensity score (negative = 0; weak = 1; moderate = 2; strong = 3) and the percentage score of immunopositive cells (no staining = 0, 1-10% of stained cells = 1; 11-50% of stained cells = 2, 51-80% of stained cells = 3; 81-100% of stained cells = 4). Each sample was scored according to these IRS criteria. An IRS of ≥6 was considered to indicate 'increased expression' of OCT-4. Figures 3A, B, C, and 3D show examples with a strong (A, B, C) and an absent (D) staining intensity, respectively.

Categorical variables were analyzed by x²-test. Multiple comparisons were corrected using Bonferroni's correction. OCT-4 mRNA and protein expression were correlated using Pearson's correlation coefficient. A univariate regression model was used to assess the influence of day of the menstrual cycle, using 3-day intervals, on OCT-4 protein expression, comparing an IRS <6 vs. ≥ 6. All p-values were two-tailed and 95% CI were calculated. A p-value < 0.05 was considered statistically significant. We used the statistical software SPSS 11.0 for Windows (SPSS Inc., Chicago, IL) for statistical analyses.