{"paper_id":"c995e223-d931-4cdd-9e81-dea97c2961ec","body_text":"Endometriosis is a common gynecological disease\naffecting 6 to 10% of women of reproductive\nage. Of these, 50 to 60% experience pelvic pain,\nand up to 50% of affected women experience infertility\n( 1 ,  2 ). Dysmenorrhea and infertility are the\nmain clinical manifestations of endometriosis. Endometriosis\nis also characterized by the presence\nof growing endometrial tissue outside of the uterine\ncavity, and by invasion, cultivation, and transfer.\nInfertility and chronic abdominal pain caused\nby endometriosis seriously impact the physical\nand mental health of women of reproductive age\n( 3 ,  4 ). Ectopic endometria usually affect the ovaries\nand can cause ovarian endometriosis (OEM).\nRepeated cyclic hemorrhage can destroy ovarian\ntissue, and cause inflammation, pelvic tissue adhesion\nand distorsion of pelvic anatomy.\nAutophagy is a form of programmed cell death,\ndistinct from apoptosis, that has special morphological\nchanges and a unique regulatory pathway.\nFor example, the autophagic body is not dependent on cysteine-aspartic protease (caspase). Autophagy\nplays a very important role in the process\nof cell growth, differentiation, tissue remodeling,\ncell immunity, environmental adaptation and\ndeath. The features of autophagic programmed cell\ndeath include double-layered autophagosome and\nmultilayered membrane-bound bubble structures\nthat encase massive amounts of cytoplasm and a\nnumber of organelles. The plasmin system eventually\nremoves these components ( 5 ,  6 ).\nBeclin-1, the mammalian homologous gene of\nyeast Atg6, has a central role in autophagy ( 7 ). The\nexpression of the Beclin-1 protein was found to be\nvery low in a variety of tumor cells. Liang et al. found\nthat the level of Beclin-1 in breast carcinoma was\nsignificantly lower than in normal breast epithelial\ntissues; the transfection of Beclin-l to human breast\ncancer cell line MCF7 inhibited  in vitro  proliferation\nand tumorigenicity of these cells, suggesting that the\ndecline of cell autophagic activity may be associated\nwith the initiation and development of tumors ( 8 ).\nEctopic endometrial cells from endometriosis patients\nhave similar biological characteristics to those\nof cancer cells. These include invasion, implantation\nand transfer. Also, both endometriosis and cancer\npatients are subject to relapse. Both endometriosis\nand adenomyosis are characterized by the presence\nof ectopic endometria outside the uterus and in the\nmyometrial wall. Recent morphologic studies have\ndemonstrated that in both endometriosis and adenomyosis,\nthe eutopic endometrium and the inner\nmyometrium show functional and structural abnormalities\n( 9 ). Studies have also shown that Beclin-1\nmRNA and protein expression are significantly lower\nin the eutopic endometrium of women with adenomyosis\nthan in healthy women. Furthermore, Beclin-\n1 has been found to be negatively correlated with\nserum cancer antigen 125 (CA125) and pelvic pain in\npatients with adenomyosis ( 10 ). However, no studies\non the expression of Beclin-1 in endometriosis have\nyet been published.\nCA125 is a cell surface antigen and a member\nof the mucin family of glycoproteins, encoded by\nMUC-16. It was discovered in the early 1980s.\nImmunocytochemical-based studies have demonstrated\nthe presence of CA125 on the surface of\ncells in endometriotic lesions ( 11 ). CA125 is a\nwell-known biomarker of endometriosis and can\nbe helpful in daily clinical practice when endometriosis\nis suspected ( 12 ).\nThe purpose of the present study is to compare\nthe expression levels of Beclin-1 mRNA and\nprotein in uterine endometria of women without\nendometriosis to those in eutopic and ectopic\nendometria of endometriosis patients using Real\ntime-PCR and Western blot analysis, and to\nevaluate the association between Beclin-1 protein\nexpression and serum CA125 levels in the\nendometriosis group.\n\nThe experimental study was approved by the Institute\nResearch Medical Ethics Committee of Anhui\nMedical University and written informed consent for\nparticipation in the study was provided by each participant.\nA total of 52 patients were selected using the\nfollowing inclusion criteria: reproductive age (18-\n40 years). Proliferative phase of menstrual cycle,no\nmedical treatments during 3 months before operation\nand no uterine devices. All patients were evaluated\nin days 3-7 after menstruation. The patients were\ndivided into an endometriosis group and a normal\nendometrial tissue group. The endometriosis group\n(experimental group) comprised of patients who had\nunilateral or bilateral ovarian chocolate cysts with diameters\n≥3 cm. 26 samples of eutopic endometrium\nand ectopic ovarian endometrial tissue were collected\nfrom patients undergoing laparoscopy or laparotomy\nwith chocolate cyst tissue confirmed by pathology.\nThe proliferative phase was confirmed by pathological\ndiagnosis. Normal endometrial tissue group\n(control group) consisted of endometrium samples\ncollected from 26 sterile women with menstrual\nregularity whose infertility was attributed to tubal\nfactor. Endometriosis was excluded by laparoscopy.\nSamples were collected during laparoscopy or pelvic\nexam and confirmed by pathological examination.\nEndometrial cell sampler (FEM103000, Unimax\nmedical systems Inc., Taiwan) was used to draw the\neutopic proliferative endometria from endometriosis\npatients and the controls.\nPatient information including age, body mass\nindex (BMI), serum CA125 level, previous pregnancy\nrate (number of previously pregnant women\ndivided by 26) and previous dilatation and curettage\nrate (number of women who underwent dilatation\nand curettage divided by 26) was collected\nfrom the patients’ clinical records and laboratory\nexamination ( Table 1 ).\nComparison of demographic and Beclin-1 expression data between endometriosis and control groups\nPatient demographic data including age, body mass index (BMI), previous pregnancy rate and previous dilatation and curettage rate showed no obvious difference. Serum CA125 level was elevated in the endometriosis group. The normalized expression level of Beclin-1 mRNA in the experimental group was significantly lower than in the control group (p<0.05). The expression level of Beclin-1 protein in the experimental group was significantly lower than in the control group (p<0.05).\nBlood samples for CA125 were taken prior to\nsurgery, centrifuged and assayed in accordance\nwith the manufacturer’s instructions. Electrochemiluminescent\nimmunoassay was used to determine\nCA125 serum levels in both groups. The\nCA125 kit and the automatic immunoassay analyzer\nwere manufactured by Abbott Laboratories,\nUSA. Normal levels of CA125 were considered\nless than 35 U/mL.\nThe grinding device was cleaned using standard\nmethods and rinsed four times with sterilized double-\ndistilled water. High-pressure disinfection was\nused to remove diethypyrocarbonate (DEPC). The\ndevice was then dried for 1 hour at 200˚C. After\nDEPC was added to the solution to a concentration\nof 0.1% and the mixture was subjected to magnetic\nstirring for 20 minutes at room temperature. It was\nthen heated to 70˚C for 1 hour to remove residual\nDEPC. The plastic products were soaked in 0.1%\nDEPC overnight, dried, and autoclaved for 15-30\nminutes.\nTotal RNA was extracted (100 mg of tissue) using\nTRIzol reagent (Invitrogen Life Technologies,\nPaisley, U.K.) according to the manufacturer’s\ninstructions. RNA was stored at -80˚C for future\nprocedures.\nAccording to the manufacturer’s instructions,\ncDNA was synthesized using AMV Reverse Transcriptase\n(Promega Biotech, Beijing, China) starting\nwith 1 μg of mRNA.\nThis process began with a 5-minute denaturation\nat 94˚C followed by 30 cycles of 30 seconds of\ndenaturation at 94˚C, 30 seconds of annealing at\n57˚C and 30 seconds of extension at 72˚C. This\nwas followed by a 10-minute extension at 72˚C.\nProtein samples were separated on a 1% agarose\ngel under denaturing conditions and electrophoretically\ntransferred to nitrocellulose\nmembranes. The membranes were probed with\n1:500 dilution of Beclin-1antibody (sc-11427,\nSanta Cruz Biotechnology, Inc., Santa Cruz, CA,\nU.S.) and then incubated with a secondary antibody\n(anti-mouse IgG, 1:5000; Beyotime Biotech,\nShanghai, China). For detection, enhanced\nchemiluminescence was carried out with BCA\nkits (Beyotime Biotech, Shanghai, China). An\ninternal reference sample (the same one for each\nblot) was included as a standard for quantification.\nThe signal from each band was measured\nagainst the standard, and this relative number\nwas used for statistical analysis.\nStatistical analysis was performed using SPSS\n19.0 statistical software package (SPSS Inc., Chicago,\nIL). Data were expressed as mean ± standard\ndeviation (SD) or percentages as appropriate.\nA single-sample Kolmogorov-Simirnov test was\nused to test normality and an independent t test\nwas used for quantitative variables. The ÷2 test was\nused for qualitative variables. ANOVA was used\nfor comparing multiple groups and the Q test was\nused to contrast between two groups. P<0.05 was\nconsidered statistically significant.\n\nThe mean ages of women with and without\nendometriosis were 34.28 ± 4.33 and 32.12 ±\n4.42 years respectively with no significant difference\nbetween them (p=0.09). There was no\ndifference in BMI, number of previous pregnancy\nrate and previous dilatation curettage\nrate. Serum levels of CA125 were significantly\nhigher in the endometriosis group than in the\ncontrol group ( Table 1 ).\nSemi-quantitative expression of Beclin-1 mRNA\nin the experimental group samples (paired eutopic\nand ectopic endometrial samples from the same\npatients) and the samples from the control group\nwas detected using RT-PCR. The normalized expression\nlevel of Beclin-1 mRNA in the experimental\ngroup was significantly lower than in the\ncontrol group (p<0.05) ( Table 1 ,  Fig 1 ). The grey\nvalue was analyzed using Jetta 801 gel imaging\nsoftware.\nSemi-quantitative expression of Beclin-1 mRNA in the experimental group (paired eutopic and ectopic endometrial\nsamples from the same patients) and the samples from the control group was detected using RT-PCR. The grey value was\nanalyzed using the Jetta 801 gel imaging software.\nE1; Endometriosis ectopic group, C; Control group and E2; Endometriosis eutopic group.\nThe expression of Beclin-1 protein was analyzed in\nthe experimental (in eutopic and ectopic endometrium\ngroup) and control groups by Western blotting. The\nexpression level of Beclin-1 protein in the experimental\ngroup was significantly lower than in the control\ngroup (p<0.05) ( Table 1 ,  Fig 2 ). The protein bands of\nβ-actin in these groups were obvious and consistent.\nDifference in Beclin-l protein levels was observed\namong the three groups of endometrial tissue.\nSerum levels of CA125 ranged from 6.98 to\n201.7 U/ ml. Pearson’s correlation coefficient (r)\nof CA125 level and Beclin-1 protein expression\nlevel in eutopic endometria of the experimental\ngroup was -0.566. This negative correlation between\nthe serum CA125 level and Beclin-1 protein\nexpression level was statistically significant\n(p=0.003) ( Fig 3 ).\nBeclin-1 protein expression presented with representative immunoblots. The protein bands of β-actin in these groups were consistent.\nE1; Endometriosis ectopic group, C; Control group and E2; Endometriosis eutopic group.\nThere was a negative correlation between Beclin-1 protein expression and serum CA125 (n=26, r=-0.566, p=0.003). The\nline refers to the changing trend of data.\n\nThis study is the first to reveal that the expression\nof Beclin-1 mRNA and protein is significantly decreased\nin ectopic and eutopic endometrium from\npatients with endometriosis. In contrast, serum\nCA125 levels were significantly higher in the endometriosis\ngroup. Beclin-1 protein expression of\neutopic endometria in patients with endometriosis\nwas negatively correlated with serum CA125 level.\nSince Beclin-1 is an autophagy-related protein,\nit was deduced that the ability of cells to undergo\nautophagy was reduced in ectopic and eutopic endometria\nof patients with endometriosis, and that\nautophagy might be related to the pathogenesis\nand progression of endometriosis.\nTheories concerning the etiology of endometriosis\ninclude the implantation hypothesis of retrograde\nmenstruation proposed by Sampson in 1927,\nthe doctrine of hematogenous and lymphatic dissemination,\ncoelomic epithelium metaplasia theory,\nimmune theory and the theory of heredity and\nenvironment. Studies have shown that endometrial\nfragments (glandular epithelial and stromal cells)\nmust undergo the adhesion, invasion and angiogenesis\nprocess so that they can grow, and cause\nlesions and clinical symptoms. However, this\nhypothesis cannot completely explain why retrograde\nmenstruation does not causeendometriosis\nin all women, indicating that other factors may\nplay important roles in the pathogenesis of endometriosis.\nEutopic endometria may play a role in\nthe initiation and progression of endometriosis and\nit is thought to be the source of lesions ( 13 ). The\nso-called eutopic endometrium determinism was\nused to revise and modify Sampson’s hypothesis.\nAlthough the eutopic endometria of women with\nand without endometriosis are histologically similar,\nstudies have shown that invasive properties,\ndecreased apoptosis, alterations in expression of\nspecific genes and proteins, and increased steroid\nand cytokine production have been observed in the\neutopic endometria of women with endometriosis.\nAlso,significant biochemical differences exist\neven between ectopic and autologous eutopic endometria\n( 14 ).\nThe Beclin-1 gene is essential to autophagy. Beclin-\n1 is located on human chromosome 17q21. It\nis 150 kb in length, and is one of about 12 genes\nin the 400 kb region ( 15 ). Liang et al. found the\nstructure of Beclin-1 to be similar to that of the\nyeast autophagy gene apg6/ vps30 and that Beclin-\n1 is part of the Bcl-2 protein family ( 8 ). One\nstudy on sequence and structure of this gene indicated\nthat Beclin-1 contains a conserved BH3\ndomain, which is both necessary and sufficient for\nits interaction with Bcl-X (L) ( 16 ). The present\nwork showed that expression of Beclin-1 mRNA\nand protein was lower in both eutopic and ectopic\nendometrium of patients with endometriosis than\nin the control group. Decreased expression of Beclin-\n1 has been found to be inversely correlated\nwith altered expression of Bcl-xL in ovarian carcinoma,\nand its expression has thus been used to\npredict patient survival in ovarian carcinomas with\nincreased expression of Bcl-xL ( 17 ). Sun et al. reported\nthat Beclin-1 protein was up-regulated in\noverexpressed transfectants of CaSki cells in cervical\ncancer ( 18 ). Other studies have shown the\npossibility that members of the Bcl-2 family may\nfunction as oncogenes not only by blocking apoptosis\nbut also by blocking autophagy ( 19 ).\nTumors were one of the first conditions to be\nfound related to autophagy. It has been confirmed\nthat autophagy is related to precancerous lesions\ninvolving carcinoma cells. Removal of Beclin-1\ngene from mouse embryonic stem cells showed\nthat beclin-1-/- mutant mice die early during embryogenesis\nand that beclin-1+/- mutant mice suffer\nfrom a high incidence of spontaneous tumors\n( 20 ). Beclin-1 is deleted in 40- 75% of cases of human\nsporadic breast, ovarian, and prostate cancers.\nKnocking down ATG5 and beclin-1, two known\nessential autophagic molecules, can cause radiation\nresistance among lung cancer cells. Therefore,\nreinforcement of autophagy may improve the effects\nof radiation therapy on lung cancer cells ( 21 ).\nResults showed that the decreased autophagic activity\nobserved in ectopic and eutopic endometrial\ncells can lead to less autophagy-dependent degradation\nof proteins and less programmed cell death.\nIn this way, ectopic endometrial cells can adhere to\nthe extracellular matrix, invade other tissues, grow\noutside the endometrium and thus cause endometriosis.\nThe correlation between levels of expression\nand the concentration of interactive sites between\nBeclin-1 and members of the Bcl-2 family\nsuggest that Beclin-1 may be involved in apoptotic\nprocesses that also involve the Bcl-2 family. The\ninactivation of Beclin-1 results in loss of control of certain genes in the Bcl-2 family and can decrease\ncapacity for autophagy in cells, allowing ectopic\nendometrial cells to survive.\nThe result of the present study showed that concentrations\nof CA 125 were higher in patients with\nendometriosis than in the control group. These results\nare similar to those found by Ramos et al. ( 22 ). CA\n125 is expressed in the eutopic and ectopic endometrium.\nThe special function of oligosaccharides\nlinked to CA125 may induce immunomodulatory\neffects and CA 125 may potently suppress the cytotoxicity\nof human natural killer (NK) cells ( 23 ,  24 ),\nthereby promoting progression of the disease. Serum\nCA 125 levels are non-surgical tools for diagnosing\nand staging pelvic endometriosis, and the value\nof serum CA 125 measurement as a diagnostic aid\nin moderate-severe stages is well recognized ( 25 ).\nMaiorana et al. showed that the mean serum CA 125\nlevels were higher during stage IV than during other\nstages of endometriosis according to criteria issued\nby the American Fertility Society (R-AFS) ( 26 ).\n\nResults suggest that Beclin-1 is associated\nwith the occurrence and development of endometriosis\nand is negatively correlated with\nCA125 serum level. These provide novel insight\ninto the pathogenesis of endometriosis. However,\nthe present experiment was not sufficiently\nthorough. Further experiments on populations\nwith larger sample may further substantiate the\nassociation between autophagy and endometriosis.\nFurther investigation should focus on the\nrelationship between the expression of Beclin-l\nin endometrial tissues and clinical symptoms.","source_license":"CC0","license_restricted":false}