{"paper_id":"5c73fb5e-fad2-453c-b4ae-a52f39252105","body_text":"Endometriosis (EM) is the growth of cells similar\nto those inside of the uterus (endometrial cells),\nbut in a location outside of the uterus ( 1 ). It is one\nof the most common benign diseases among women\n( 2 ). However, its pathologic mechanism still\nremains unclear. There are differences in gene expression\nbetween endometriotic tissue and eutopic\nendometrium ( 3 ,  4 ) and the characteristics of the\nEM endometrium and its growth environment play\nimportant roles in the initiation and development\nof EM ( 5 ,  6 ). Growth hormone-releasing hormone\n(GHRH) is a polypeptide hormone containing 42-\n44 amino acids. Originally, it was thought that\nGHRH was the product of hypothalamic secretion,\nand exerted its functions in the pituitary gland to\nactivate the synthesis and secretion of growth hormone\nand regulate the proliferation and differentiation of pituitary somatotropes. But recent studies\nshow that GHRH is expressed in other tissues\nin addition to the pituitary, suggesting that GHRH\nhas extensive biological effects ( 7 ).\nGHRH receptors contain seven transmembrane\ndomains. They have a relatively high degree\nof homology with receptors such as vasoactive\nintestinal peptide, pituitary adenylate\ncyclase activating peptide and calcitonin.\nTherefore, GHRH can exert its extra-pituitary\nfunctions via different receptors according to\ndifferent types of cells ( 8 ). It plays its roles as\nan autocrine growth factor in many types of tumors.\nUp to now, some extra-pituitary GHRH\nreceptors have been identified and their splice\nvariant cDNA has been detected in tumor tissues\n( 9 - 11 ). GHRH-SV1, a splice variant of\nGHRH receptors, is highly similar to the pituitary\nGHRH receptor and can mediate and\npromote mitosis. It exerts its biologic effects\nthrough its integration with GHRH. The integration\nactivates adenylate cyclase to produce\ncyclic adenosine monohosphate (cAMP) which\nis the common second signal of the GHRH receptors\n( 12 - 14 ).\nPrevious study showed that GHRH is expressed\nin eutopic endometrium ( 15 ).This study explores\nwhether GHRH and GHRH-SV1 are expressed\nin eutopic endometrium and endometriotic tissue,\nand analyzes the possible differences in their expression\nat different clinical stages of EM.\n\nIn this research paper, 80 EM patients were\ninvolved in the current study, whose age ranged\nfrom 22 to 48 years (35.5 ± 2.0). They were diagnosed\nwith EM by laparoscopy or pathology after\nopening surgery in Ningbo Women and Children’s\nHospital between March 2009 and September\n2010. Among all subjects, 20 were at stages I, II,\nIII and IV. The specimens were taken from ectopic\nendometrium and endometriotic tissue during operation.\nThe control group was comprised of 50\nnon-EM patients who underwent hysterectomy\nbecause of myoma during the same period, with\nage range of 20-49 (mean 35.0 ± 2.5) years. The\nage range showed no significant difference compared\nwith the experiment group (p>0.05). All\nspecimens were not infected. All enrolled patients\nhad regular menstrual cycles without internal complications,\nsuch as diabetes, high blood pressure,\nheart disease and endocrine system disease. All\npatients had no other endometrial diseases such\nas endometrial polyps, uterus gland myopathy and\nthe merger reproductive system malignant tumors.\nThey didn’t receive hormonal therapy within\nthree months before the operation. EM staging was\nin accordance with the revised American Society\nfor Reproductive Medicine (rASRM).The stage\nand score of each patient were performed by one\nchief physician and two attending physician who\nwere involved in the operation. This study was\nconducted with approval from the Ethics Committee\nof Ningbo Women & Children’s Hospital, China.\nWritten informed consent was obtained from\nall participants.\nEach sample was divided into two fragments.\nOne fragment was washed with saline, fixed in\n10% formaldehyde and embedded in paraffin. Serial\nsections were then prepared at the thickness\nof 4 μm. The other was put into an eppendorf tube\nafter washing with saline which was further placed\ninto an ice cylinder, and then sent to the lab immediately\nfor storage at -80˚C.\nThe procedures were performed according to the\ninstructions indicated in the streptavidin-peroxidase\n(SP) kit (Beijing Zhongshan Biotechnology\nCo., LTD, China). After staining, slice with the\nbrown particles represented that the tissue contained\nthe detected material. The sections were\nanalyzed using the image processing system and\nHPIAS-1000 high-resolution image analyzing\nsoftware to determine the staining intensity and\ndistribution range. Five amplified fields (40×10)\nwere selected randomly and the mean optical density\n(OD) value in each field was measured for\nquantitative analysis.\nTissue (0.1 g) was grinded into pulp and 1ml\nTrizol reagent (Invertrogen, USA) was added\nfor total RNA extraction according to the\nmanufacturer’s instructions. The absorbance of 260/280 was measured to calculate the concentration\nand purity of the RNA sample. Samples with\na 260/280 ratio between 1.8 and 2.0 were taken for\nRT-PCR detection.\nThe procedures were performed according to the\ninstructions indicated in the RT-PCR kit (Beijing\nZhongshan Biotechnology Co., Ltd., China). The\namplification conditions for GHRH included an\ninitial pre-denaturation at 95˚C for 10 minutes followed\nby 40 cycles of 94˚C for 30s, 60˚C for 30\ns and 72˚C for 1 minute. Glyceraldehyde-3-phosphate\ndehydrogenase (GAPDH) was used as an\ninternal control. The amplification conditions for\nGHRH- SV1 included an initial pre-denaturation\nat 95˚C for 3 minutes followed by 35 cycles of\n95˚C for 30s, 58˚C for 30s and 72˚C for 2 minutes.\nThe forward and reverse primers are listed\nin table 1. PCR products (5 μl) were analyzed in\n1.5% agarose gel electrophoresis and results were\nobserved under the ultraviolet projection reflector.\nDNA Marker (TakaRa Co., LTD, China) was used\nas the DNA length marker. Dot intensity scanning\nwas performed for positive straps using the digital\nimaging system and GAPDH correction was carried\nout for relative amount analyses. The expression\nintensity of a target gene was determined by\nthe ratio between the absorbance of the target gene\nproducts and that of the GAPDH products.\nData were presented by means ± standard error\n(Mean ± SEM), and analyzed using the SPSS 12.0\nsoftware. Analysis of Variance (ANOVA) with\nTukey Post Hoc test was carried out and p<0.05\nwas considered statistically significant. All data\nwere normally distributed and had homogeneous\nvariances.\n\nGHRH, GHRH-SV1 and their corresponding\nmRNA were expressed in eutopic endometrium and\nendometriotic tissue as well as ectopic endometrium.\nThe mean OD values of GHRH and GHRHSV1\nin the experimental group were significantly\nhigher than those in the control group (p<0.05). The\nRI of GHRH and GHRH-SV1 mRNA in the experimental\ngroup were significantly higher than those\nin the control group ( p<0.05) ( Table 2 ,  Fig 1A, B ).\nThe mean OD values of GHRH and GHRH-SV1 in\nectopic endometrium were significantly higher than\nthose in endometriotic tissue (p<0.05).\nThe primer sequence used in RT-PCR\nExpression of GHRH, GHRH-SV1 and the corresponding mRNA in eutopic endometrium and endometriotic tissue\nA. Electrophoresis of expression of GHRH, GHRHSV1\nand the corresponding mRNA. Lane M; DNA marker,\nLane N; Negative control, Lane 1; GHRH (322bp) and\nGAPDH (226bp), Lane 2; GHRH-SV1 (720bp) and GAPDH\n(226bp)and Lane 3; GAPDH (internal control 226bp).\nB. Analysis of expression of GHRH, GHRH-SV1 and the\ncorresponding mRNA in normal endometrium and endometriosis\ntissues (Y axis represents the corresponding GHRH,\nGHRH-SV1 and mRNA expression level).\nAs shown in table 3 and figure 2, ANOVA\nshow significant differences in GHRH and\nGHRH-SV1 expression among the different\nstages of EM. Variance analyses show significant\ndifferences in GHRH and GHRH-SV1\nmRNA expression among the different stages\nof EM (p<0.05).\nExpression of GHRH, GHRH- SV1 and the corresponding\nmRNA in different stages of endometriosis lesions\n(Y axis represents the corresponding GHRH and GHRHSV1\nmRNA expression level).\nExpression of GHRH, GHRH- SV1 and the corresponding mRNA in different stages of endometriosis lesions\n\nResearch has found that GHRH can play roles\nbesides of the pituitary tissues and SV1 displays\nthe closest sequence similarity to GHRH receptors\namong various splice variants which can express\nGHRH receptors in human tumors ( 16 ). It is\na type of functional receptor which can induce the\nmitosis in tumors as well as other GHRH-related\nsignals ( 17 ,  18 ). Positive-stained GHRH receptors\nare found in the human endometrium ( 19 ). This research\nprompts that GHRH and the splice variants\nof GHRH receptors may have important roles in the\npathogenesis of EM.\nOur study shows that GHRH and GHRH-SV1\nare expressed in eutopic endometrium and endometriotic\ntissue as well as ectopic endometrium.\nThe GHRH and SV1 expression in endometriotic\ntissue is significantly higher than that in eutopic endometrium. In this study, we find that the GHRH\nand SV1 expression in ectopic endometrium is\nsignificantly higher than that in endometriotic\ntissue. Different stages of EM also show significant\ndifferences in GHRH and SV1 expression.\nRecently, Fu et al. ( 20 ) found when ectopic endometrial\nstromal cells (ESC) were isolated and\ncultured with growth hormone-releasing hormone,\nthe production of cAMP and the incorporation of\n5-bromo-2'-deoxyuridine in SV1-expressing ESC\nis stimulated. These results suggest that the interaction\nbetween GHRH and SV1 may be a possible\nmechanism in the initiation and development\nof EM. In this study, the differences in the GHRH\nand SV1 expression among different stages of\nEM exhibit a non-linear relationship between the\nstages and GHRH and SV1 expression ( Fig 2 ),\nsuggesting that other signaling pathways may also\nplay roles in the process.\nTreatment of endometriosis is a difficult matter.\nThe finding on GHRH and SV1 will represent a\nnew approach. Annunziata et al. found that the\nGHRH antagonist JV-1-36 inhibited endometriotic\ncell proliferation and survival, suggesting that the\nGHRH antagonist may represent a promising tool\nfor treatment of endometriosis ( 21 ).\n\nTo sum up, the actual mechanism underlying EM\nstill remains unclear. The current study is expected to\nprovide a possible explanation for the pathogenesis\nof EM as well as an option in its treatment.","source_license":"CC0","license_restricted":false}