Comparison of Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variant 1 in Different Stages of Endometriosis

In this research paper, 80 EM patients were involved in the current study, whose age ranged from 22 to 48 years (35.5 ± 2.0). They were diagnosed with EM by laparoscopy or pathology after opening surgery in Ningbo Women and Children’s Hospital between March 2009 and September 2010. Among all subjects, 20 were at stages I, II, III and IV. The specimens were taken from ectopic endometrium and endometriotic tissue during operation. The control group was comprised of 50 non-EM patients who underwent hysterectomy because of myoma during the same period, with age range of 20-49 (mean 35.0 ± 2.5) years. The age range showed no significant difference compared with the experiment group (p>0.05). All specimens were not infected. All enrolled patients had regular menstrual cycles without internal complications, such as diabetes, high blood pressure, heart disease and endocrine system disease. All patients had no other endometrial diseases such as endometrial polyps, uterus gland myopathy and the merger reproductive system malignant tumors.

They didn’t receive hormonal therapy within three months before the operation. EM staging was in accordance with the revised American Society for Reproductive Medicine (rASRM).The stage and score of each patient were performed by one chief physician and two attending physician who were involved in the operation. This study was conducted with approval from the Ethics Committee of Ningbo Women & Children’s Hospital, China. Written informed consent was obtained from all participants.

Each sample was divided into two fragments. One fragment was washed with saline, fixed in 10% formaldehyde and embedded in paraffin. Serial sections were then prepared at the thickness of 4 μm. The other was put into an eppendorf tube after washing with saline which was further placed into an ice cylinder, and then sent to the lab immediately for storage at -80˚C.

The procedures were performed according to the instructions indicated in the streptavidin-peroxidase (SP) kit (Beijing Zhongshan Biotechnology Co., LTD, China). After staining, slice with the brown particles represented that the tissue contained the detected material. The sections were analyzed using the image processing system and HPIAS-1000 high-resolution image analyzing software to determine the staining intensity and distribution range. Five amplified fields (40×10) were selected randomly and the mean optical density (OD) value in each field was measured for quantitative analysis.

Tissue (0.1 g) was grinded into pulp and 1ml Trizol reagent (Invertrogen, USA) was added for total RNA extraction according to the manufacturer’s instructions. The absorbance of 260/280 was measured to calculate the concentration and purity of the RNA sample. Samples with a 260/280 ratio between 1.8 and 2.0 were taken for RT-PCR detection.

The procedures were performed according to the instructions indicated in the RT-PCR kit (Beijing Zhongshan Biotechnology Co., Ltd., China). The amplification conditions for GHRH included an initial pre-denaturation at 95˚C for 10 minutes followed by 40 cycles of 94˚C for 30s, 60˚C for 30 s and 72˚C for 1 minute. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The amplification conditions for GHRH- SV1 included an initial pre-denaturation at 95˚C for 3 minutes followed by 35 cycles of 95˚C for 30s, 58˚C for 30s and 72˚C for 2 minutes. The forward and reverse primers are listed in table 1. PCR products (5 μl) were analyzed in 1.5% agarose gel electrophoresis and results were observed under the ultraviolet projection reflector. DNA Marker (TakaRa Co., LTD, China) was used as the DNA length marker. Dot intensity scanning was performed for positive straps using the digital imaging system and GAPDH correction was carried out for relative amount analyses. The expression intensity of a target gene was determined by the ratio between the absorbance of the target gene products and that of the GAPDH products.

Data were presented by means ± standard error (Mean ± SEM), and analyzed using the SPSS 12.0 software. Analysis of Variance (ANOVA) with Tukey Post Hoc test was carried out and p<0.05 was considered statistically significant. All data were normally distributed and had homogeneous variances.

GHRH, GHRH-SV1 and their corresponding mRNA were expressed in eutopic endometrium and endometriotic tissue as well as ectopic endometrium. The mean OD values of GHRH and GHRHSV1 in the experimental group were significantly higher than those in the control group (p<0.05). The RI of GHRH and GHRH-SV1 mRNA in the experimental group were significantly higher than those in the control group ( p<0.05) (Table 2, Fig 1A, B). The mean OD values of GHRH and GHRH-SV1 in ectopic endometrium were significantly higher than those in endometriotic tissue (p<0.05).

As shown in table 3 and figure 2, ANOVA show significant differences in GHRH and GHRH-SV1 expression among the different stages of EM. Variance analyses show significant differences in GHRH and GHRH-SV1 mRNA expression among the different stages of EM (p<0.05).