卵巢子宫内膜异位症的输卵管上皮细胞起源研究
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摘要
背景和目的：子宫内膜异位症是一种常见的妇科良性疾病,它是子宫内膜组织在子宫腔以外的部位生长而引起的疾病,卵巢是最常受累的器官。异位内膜组织由子宫内膜样腺体和间质组成,具有功能活性,在局部呈浸润性生长和反复出血,引起相应的组织学改变和临床症状,患者通常表现为慢性盆腔痛,性交痛,出现月经异常,甚至造成不孕症。据统计大约有10%的育龄期女性会罹患子宫内膜异位症,并且高达50%的子宫内膜异位症患者因此正遭受慢性腹痛和不孕症的折磨。尽管100多年前Carl von Rokitansky医生首次辨明并详细记载了子宫内膜异位症,从此人们对该病开始有所认识并为之进行了许多开拓性研究,但是到目前为止,人们对子宫内膜异位症的发病机制还不是十分清楚。目前关于子宫内膜异位症的发生学说,为大家所普遍接受的是被称为“子宫内膜异位症之父”的Sampson医生于20世纪20年代提出的经血逆流种植学说。但是该学说依然存在许多矛盾的地方,比如有将近90%的育龄期女性存在经血逆流的现象,然而只有5%～10%的育龄期女性发现患有子宫内膜异位症。经血逆流学说可以用来解释发生在盆腹腔内的子宫内膜异位症,但是该学说尚不能解释出现在腹腔以外的子宫内膜异位症。经血逆流学说的这些矛盾之处使得Iwanoff及同时期的Meyer医生提出的体腔上皮化生学说找到了用武之地,体腔上皮化生学说的核心是认为子宫内膜异位症是由间皮细胞经过化生过程转化而来。在腹腔以外的远隔部位发生的子宫内膜异位症以及在男性人群中鲜见的子宫内膜异位症则支持这种理论学说。尽管体腔上皮化生学说能够圆满解释大多数的卵巢上及腹腔内的子宫内膜异位症,但是该学说缺少细胞或者分子水平的证据支持。与此同时,子宫内膜碎片经淋巴管道以及血管途径转移到远处也被视作为子宫内膜异位症发生的一个补充理论,它常常能够用来解释出现在远离盆腔的罕见部位出现的子宫内膜异位症,但是该理论尚不能作为子宫内膜异位症播散种植的主要发生机制。近年来兴起的干细胞学说则认为来源于骨髓的干细胞可以在盆腔内外分化成为子宫内膜样组织,然而这个干细胞理论毕竟是刚刚起步,尚未完全清晰明了。综上所述,目前为止尚无单一理论学说能够完美的解释任何位置的子宫内膜异位症的发病机制。
许多年以来,女性输卵管仅仅被视为含子宫内膜碎片的经血逆流进入盆腔以及输送卵子及受精卵进入子宫腔着床的一个肌性通道。人们从来没有怀疑输卵管可能会是子宫内膜异位症的一种重要的组织细胞起源。我们在临床上通过对大量病患的输卵管病理切片的检视,发现输卵管可能在某种程度上参与了卵巢子宫内膜异位症的形成。我们经常能在行输卵管结扎术的组织标本的在靠近宫腔一侧的输卵管管腔中发现输卵管粘膜上皮子宫内膜样变,并且我们目前发现输卵管的粘膜上皮能够形成子宫内膜样的组织。许多能存活的上皮细胞能够从输卵管粘膜脱落下来在卵巢表面形成输卵管内膜异位(endo salpingiosis)或者称为卵巢表面上皮包涵体,在大约30%的病例中能够发现这种包涵体。近来我们在关于卵巢低级别浆液性癌的细胞起源的研究中发现大部分卵巢表面上皮包涵体来源于输卵管粘膜上皮,此种卵巢包涵体很可能通过化生过程转化为卵巢子宫内膜异位症。
基于以上各种学说以及临床病理发现,我们大胆推测输卵管粘膜上皮参与了卵巢子宫内膜异位症形成。为了验证我们的推测,必须找到一些特异性的生物标记物,能够将卵巢子宫内膜异位症区分出是输卵管细胞起源还是来源于子宫内膜。因此在本研究中,我们通过全基因组表达谱芯片技术筛选出一系列在输卵管粘膜上皮和在子宫内膜组织中存在明显的差异表达的基因,而后在卵巢子宫内膜异位症组织中分析筛选出的差异基因的表达状况并与配对的输卵管粘膜上皮及子宫内膜组织中的该基因的表达状况进行比较,来判断卵巢子宫内膜异位症的细胞起源。
材料与方法：收集山东大学齐鲁医院妇科2010年至2013年期间56例住院患者的共147例标本。研究对象因卵巢子宫内膜异位症或其他妇科良性疾病行全子宫加双侧附件切除术。其中35例患者患有卵巢子宫内膜异位症,术后获得输卵管伞,配对子宫内膜及卵巢子宫内膜异位症组织。21例患者无子宫内膜异位症,术后获得输卵管伞及配对子宫内膜。组织标本取得后切成小块液氮速冻或经10%中性福尔马林固定后包埋便于组织学鉴别及研究。包含上皮组织和间质的卵巢子宫内膜异位症病灶组织,输卵管粘膜上皮及相应子宫内膜经显微镜下行微切割,留待后续的实时定量PCR或Western blot蛋白印迹实验。从21例非卵巢子宫内膜异位症患者组织标本中取3对配对的新鲜输卵管伞及子宫内膜组织,进行全基因组表达谱芯片实验并分析找出组织特异性的标记物。基因筛选标准：在两种组织中该基因的表达差异大于2倍且p<0.05。根据芯片结果及筛选标准,选定目的基因：FMO3和DMBTl,并在21例非卵巢子宫内膜异位症患者组织标本中通过实时定量PCR进行进一步验证。利用实时定量PCR分析35例卵巢子宫内膜异位症患者输卵管伞,子宫内膜以及卵巢内异症病灶三处组织中的目的基因表达状况。利用Western Blot蛋白印迹实验在蛋白水平上分析上述组织标本中FM03和DMBTl的表达状况。利用免疫组化技术分析FM03和DMBTl在输卵管伞,子宫内膜和卵巢子宫内膜异位症组织切片中的表达分布状况。
结果：
1.全基因组表达谱芯片分析发现多种差异表达基因在输卵管与子宫内膜中分别有4114个和3451个基因存在差异表达,共有1796个基因表达差异倍数在2倍以上。与子宫内膜比较,输卵管中共有911个上调2倍以上的基因,包括8个50倍以上的基因,28个20倍以上的基因,及875个2倍以上的基因。与输卵管伞比较,子宫内膜中共有885个上调2倍以上的基因,包括7个20倍及以上的基因,878个2倍及以上的基因,尚无上调50倍以上的基因。
2.FM03基因在输卵管伞中高表达,而DMBT1基因在子宫内膜中高表达实时定量PCR实验证实21例非卵巢子宫内膜异位症患者配对的输卵管和子宫内膜组织中2种基因的表达状况：FM03在输卵管中高表达(平均上调倍数44.38,p<0.001),而DMBT1在子宫内膜中高表达(平均上调倍数22.11,p＜0.001)。Western blot蛋白印迹实验显示FM03在输卵管伞中高表达(平均上调倍数11.05,p=0.006),而DMBT1在输卵管伞中低表达(平均下调倍数32.08,p=0.001)。免疫组化显示FM03和DMBT1为细胞浆着色。多数输卵管上皮细胞呈现FM03中等或强阳性染色。21例子宫内膜组织腺体细胞呈弥漫性DMBT1强阳性染色,输卵管上皮细胞尚未见DMBT1阳性染色。
3.FM03及DMBT1在卵巢子宫内膜异位症组织中的表达FM03及DMBT1在35例卵巢子宫内膜异位症患者的输卵管伞和子宫内膜组织中在基因转录水平及蛋白水平的表达趋势与非卵巢子宫内膜异位症患者一致。有32例患者三处组织标本均可用于实时定量PCR分析,与对应的子宫内膜比较,其中18例卵巢子宫内膜异位症组织FM03表达上调,平均上调倍数7.21倍(p=0.016)；与对应的输卵管伞比较,14例卵巢子宫内膜异位症组织标本中FM03表达明显下调,但是与对应的子宫内膜组织比较,没有统计学差异。在18例上调表达FM03的卵巢子宫内膜异位症组织标本中,与对应的子宫内膜组织比较,其DMBT1的表达明显下调,平均下调倍数6.94(p=0.022)；但是与对应输卵管伞比较,没有统计学差异(p=0.144)。Western blot蛋白印迹实验显示上述18例上调表达FM03同时下调表达DMBT1的卵巢子宫内膜异位症组织中FM03及DMBT1蛋白表达趋势同mRNA水平表达趋势,其中FM03蛋白表达平均上调7.2倍。剩余17例卵巢子宫内膜异位症组织标本Western blot蛋白印迹实验分析显示8例与子宫内膜中具有相似的FM03蛋白表达水平,9例与对应子宫内膜中该蛋白表达水平无统计学差异。19例卵巢子宫内膜异位症患者三处组织Western blot蛋白印迹实验显示DMBT1蛋白在卵巢子宫内膜异位症组织中的表达状况与在输卵管伞中表达状况相似,无统计学差异,但子宫内膜组织DMBT1蛋白表达明显上调。剩余16例卵巢子宫内膜异位症患者中,与输卵管伞比较,DMBT1蛋白在卵巢子宫内膜异位症组织中表达上调,其中有12例上调表达显著,另有4例卵巢子宫内膜异位症组织与对应子宫内膜比较没有显示表达差异。35例卵巢子宫内膜异位症组织免疫组化显示,19例子宫内膜异位症上皮细胞的胞浆呈FM03中等阳性染色,剩余16例有10例局灶性阳性着色,6例因上皮组织缺如无法着色。有17例卵巢子宫内膜异位症组织切片呈现DMBT1染色阴性,剩余18例其中10例卵巢子宫内膜异位症阳性染色。
结论：近60%的卵巢子宫内膜异位症病例其子宫内膜异位症病灶组织细胞可能起源于输卵管上皮细胞,40%的卵巢子宫内膜异位症病例的子宫内膜异位症病灶组织细胞可能源自子宫内膜细胞。输卵管上皮细胞是卵巢子宫内膜异位症的一种潜在的组织细胞起源。
Background and Purpose:Endometriosis is defined by the presence of endometrial tissue outside the uterus. It is one of the most common benign gynecologic disorders associated with pelvic pain and infertility. Endometriosis, most commonly involving the ovary, affects approximately10%of women in their reproductive age and up to50%of women suffering infertility and abdominal pain. The patho genesis of endometriosis remains unclear and elusive since it was first described by Von Rokitansky over100years ago. In1920s, Sampson's retrograde menstruation theory has been widely accepted, but remains controversial as retrograde menstruation occurs in up to90%of women in reproductive age but only6%to10%of those women have endometriosis. Retrograde menstruation may explain occurrence of endometriosis within the pelvis or abdominal cavity but fails to explain the presence of endometriosis in remote sites outside the peritoneal cavity. These controversial points led Iwanoff and Meyer to propose the coelomic metaplasia theory, which explained that endometriosis may derive from mesothelial cells through metaplasia. The presence of endometriosis in remote areas and the rare endometriosis in males support the metaplasia theory. Although this metaplastic theory may explain the majority of endometriosis both in and outside of the ovary and within the peritoneal cavity, it lacks a cellular or molecular basis. Lymphatic and vascular transportation of the endometrium has also been proposed as a complimentary theory to explain rare cases of endometriosis occurring in unusual locations far from the pelvis, but this is unlikely to be the primary mechanism of disease spread. Another compelling proposal suggests that bone marrow derived stem cells may differentiate into endometriotic tissue within and outside of endometrial cavity. However, the stem cell theory is premitive and not completely understood. Overall, no single theory perfectly accounts for the pathogenesis of all cases of endometriosis.
The fallopian tube was previously recognized only as a carrier for the menstrual endometrium to pass into the peritoneal cavity or the ovarian surface. The fallopian tube has never been examined as a possible tissue or cellular source of endometriosis. Based on our clinical observations of tubal specimens, we think that the fallopian tube is likely in the certain degree to contribute the formation of ovarian endometriosis. Tubal mucosa is known to be able to form endometrial-like tissue. For instance, endometrialization is commonly seen within the tubal lumen after tubal ligation. It is also known that tubal epithelia shed viable cells onto the ovarian surface forming endosalpingiosis or ovarian epithelial inclusions, a common finding seen within the ovary in approximately30%of the cases. In a recent study onthe cell origin of low-grade serous carcinoma of the ovary, we demonstrated that the majority of the OEIs are derived from tubal epithelia and the OEIs could be transformed into ovarian endometriosis through a probable metaplastic process. We hypothesize that tubal epithelium contributes to the formation of ovarian endometriosis. To examine this provocative hypothesis, we searched for biomarkers which are specific to help differentiate tubal from endometrial origin of endometriosis within the ovary. In this study, we identified a set of novel genes which are either highly expressed in the fallopian tube or in the endometrium through a gene differential array study. We validated these unique genes and their corresponding protein expression in ovarian endometriosis by comparing their expression levels to paired specimens of fallopian tube and endometrium within individual patients.
Material and Methods:Tissue samples including human tubal fimbria, paired endometrium and ovarian endometriosis were obtained from surgical pathology specimens within30minutes of resection at the Department of Gynecology, Qilu Hospital of Shandong University from2010to2013. A total of147specimens derived from56patients were studied. Among them, each of35patients with ovarian endometriosis generated a set of samples including fallopian tube, endometrium, and ovarian endometriosis. The remaining non endometriosis patients generated21paired fallopian tube and endometrial samples. All these patients underwent total hysterectomy and bilateral salpingo-oophorectomy for either ovarian endometriosis or benign gynecologic disease without endometriosis. The patients' age ranged from35to51years with a mean age of41.5. Representative portions of the same tissue specimen were either snap frozen and stored in liquid nitrogen until use or fixed in10%neutral formaldehyde overnight and embedded in paraffin for routine histological examination. Tissue sections containing areas of endometriosis, tubal mucosa and endometrium with both glandular epithelia and stroma were confirmed under microscope and hand microdissected for either real-time PCR or Western analysis. In order to identify tissue specific bio markers, we compared the gene expression between the fallopian tube and the endometrium from patients without evidence of endometriosis by whole-genome microarray analysis. Three pairs of fresh human endometrium and corresponding tubal fimbria specimens were selected from the pool of the21paired samples mentioned above, labeled, and sent to perform whole-genome expression microarray analysis. The criteria for selection of differentially expressed genes were (1) The cut-off value differentially expressed level between the tubal fimbria and the endometrium is more than2-fold;(2) p＜0.05in fold change filtering. Genes that fit these criteria were considered significant for discrimination. The target genes for this study were selected based on the level of expression after comparing with tubal expression:FMO3and DMBT1. To verify the gene expression data obtained from the microarray, real-time PCR was performed on2selected genes, using total RNA from21paired tubal fimbria and corresponding endometrial samples including those three paired specimens for the gene array analysis. Thirty-five cases with ovarian endometriosis and paired tubal and endometrial samples were analyzed by real-time PCR. All samples mentioned above were subsequently evaluated for protein expression of FMO3and DMBT1by Western blot. Immunohistochemistry (IHC) with antibodies to FMO3and DMBT1was performed on the sections of fallopian tubal mucosa, corresponding endometrium and ovarian endometriosis respectively.
Results:
1. Multiple unique genes identified in the fallopian tube over the endometrium A total of4114and3451genes were identified in the fallopian tube and endometrium, respectively. There were1796genes identified with more than2-fold differential expression between human fallopian tube and endometrial tissues. All these differentially expressed genes were further scrutinized and the highly differentially expressed genes were summarized. Compared with the endometrium, the fallopian tube showed a total911up-regulated genes. These included50-fold or more (n=8),20-fold or more (n=28), and2-fold or more (n=875). Compared with the fallopian tube, the endometrium showed a total of885up-regulated genes including20-fold or more (n=7) and2-fold or more (n=878). There were no genes with more than50-fold up-regulation found in the endometrial tissue.
2. FMO3was highly expressed in the fallopian tube, while DMBT1was in the endometrium After screening, we identified2genes, one was highly expressed in the fallopian tube (FM03) and the other in the endometrium (DMBT1), which matched our specified conditions. We validated these two genes with real-time PCR, Western blot and immunohistochemistry. Both the FMO3and DMBT1genes follow the trend differences of the microarray results. In the21pairs of the tubal and corresponding endometrial samples in real-time PCR analysis, FM03was highly expressed in the fallopian tube compared with the paired endometrium, with average fold change of44.38(p＜0.001). In contrast, DMBT1was highly expressed in endometrium compared with the fallopian tube with average fold change of22.11(p＜0.001). None of the21pairs of samples showed discordance with the trend towards co-expression of FMO3and DMBT1in fallopian tube versus endometrial tissue. Among the21pairs of tubal and endometrial samples,14pairs were adequate for Western blot analysis. FMO3protein expression was significantly higher in the fallopian tube samples than that in the endometrium, with an average fold of increment11.05(p=0.006). In contrast, the DMBT1protein level was significantly lower in the fallopian tube (average decreasing fold=32.08) compared with the expression in the endometrium (p＝0.001). These results were compatible with the findings from real-time PCR validation, indicating FMO3and DMBT1do not change significantly at the transcriptional and post-transcriptional levels. All21pairs of the tissue samples were studied for the cellular location of both FM03and DMBT1by immunohistochemistry. Both bio markers were mainly cytoplasmic. No nuclear stainings were identified for these2genes. Of the21paired samples,19(91%) showed moderately to strongly positive staining of FM03in the majority epithelial cells of the fallopian tube, while the remaining2tubal samples were weakly stained. In contrast, FM03was only weakly and focally expressed in the3(14%) endometrial samples, mainly within the endometrial glands. There were some stromal and endothelial cell stainings identified with no specific pattern. DMBT1was strongly and diffusely positive in the majority of glandular cells in all21endometrial samples, but not in the tubal sections we studied.
3. FMO3and DMBT1expression in ovarian endometriosis. There were a total of35patients with the requisite three samples for the study. These gene expression levels were compared in patients with ovarian endometriosis and those without ovarian endometriosis from the data presented above. Both FM03and DMBT1expression levels in the fallopian tubal and endometrial samples showed no statistical difference between the patients with ovarian endometriosis and those without ovarian endometriosis. Among the35cases with ovarian endometriosis,32paired samples were adequate for real-time PCR analysis. FMO3was highly expressed in18(56%) of the32samples studied, with the average fold increment of7.21(p=0.016) compared with the endometrium. However, FMO3expression in the remaining14ovarian endometriosis samples was significantly lower than that in fallopian tube (p<0.01), but similar to the level of expression in the corresponding endometrium (p=0.184). In contrast, the18patients with high level of FM03expression, showed a significantly low expression of DMBT1in the ovarian endometriosis lesions compared with the paired endometrial samples (average decreasing fold=6.94, p=0.022). Meanwhile, DMBT1expression showed no statistical differences between the ovarian endometriosis and fallopian tube samples (p=0.144). The18ovarian endometriosis samples with high FMO3and low DMBT1expression also showed concordant level of the protein expression by Western blot. The FM03expression increased with an average fold increment of7.2(p=0.007) compared with the expression in the endometrium. Among the remaining17ovarian endometriosis samples,8showed a similar level of FMO3expression to the endometrium but lower than the tube, while9showed no statistical difference compared to either the endometrium or the fallopian tube. Among the35paired patients,19cases showed that DMBT1protein expression in ovarian endometriosis samples was similarly low in the fallopian tube, while significantly high in the endometrium. DMBT1in the remaining16cases showed high expression in areas of ovarian endometriosis and the endometrium (n=12) and no difference (n=4) between ovarian endometriosis and paired endometrium. Cellular localization of these gene products was examined by immunohistochemistry. Among the35paired samples,19(54%) showed that FMO3was positively staining in the cytoplasm of ovarian endometriosis epithelium, while the remaining16ovarian endometriosis samples showed either low expression in focal areas (n=10) or inadequate expression for analysis due to loss of glandular epithelia within the sections (n=6). In terms of DMBT1,17(49%) ovarian endometriosis samples were negative, while the remaining18ovarian endometriosis samples showed positive staining (n=10) or were inadequate (n=8).
Conclusion:Based on our preliminary study, the findings suggest that approximately60%of the ovarian endometriosis we studied may be derived from the fallopian tube, while about40%of the cases may be of endometrial origin. The fallopian tube epithelia may represent one of the tissue sources contributing to ovarian endometriosis. Such novel findings, which require confirmation, may have a significant clinical impact in searching for alternative ways of prevention and treatment of endometriosis.
许多年以来,女性输卵管仅仅被视为含子宫内膜碎片的经血逆流进入盆腔以及输送卵子及受精卵进入子宫腔着床的一个肌性通道。人们从来没有怀疑输卵管可能会是子宫内膜异位症的一种重要的组织细胞起源。我们在临床上通过对大量病患的输卵管病理切片的检视,发现输卵管可能在某种程度上参与了卵巢子宫内膜异位症的形成。我们经常能在行输卵管结扎术的组织标本的在靠近宫腔一侧的输卵管管腔中发现输卵管粘膜上皮子宫内膜样变,并且我们目前发现输卵管的粘膜上皮能够形成子宫内膜样的组织。许多能存活的上皮细胞能够从输卵管粘膜脱落下来在卵巢表面形成输卵管内膜异位(endo salpingiosis)或者称为卵巢表面上皮包涵体,在大约30%的病例中能够发现这种包涵体。近来我们在关于卵巢低级别浆液性癌的细胞起源的研究中发现大部分卵巢表面上皮包涵体来源于输卵管粘膜上皮,此种卵巢包涵体很可能通过化生过程转化为卵巢子宫内膜异位症。
基于以上各种学说以及临床病理发现,我们大胆推测输卵管粘膜上皮参与了卵巢子宫内膜异位症形成。为了验证我们的推测,必须找到一些特异性的生物标记物,能够将卵巢子宫内膜异位症区分出是输卵管细胞起源还是来源于子宫内膜。因此在本研究中,我们通过全基因组表达谱芯片技术筛选出一系列在输卵管粘膜上皮和在子宫内膜组织中存在明显的差异表达的基因,而后在卵巢子宫内膜异位症组织中分析筛选出的差异基因的表达状况并与配对的输卵管粘膜上皮及子宫内膜组织中的该基因的表达状况进行比较,来判断卵巢子宫内膜异位症的细胞起源。
材料与方法：收集山东大学齐鲁医院妇科2010年至2013年期间56例住院患者的共147例标本。研究对象因卵巢子宫内膜异位症或其他妇科良性疾病行全子宫加双侧附件切除术。其中35例患者患有卵巢子宫内膜异位症,术后获得输卵管伞,配对子宫内膜及卵巢子宫内膜异位症组织。21例患者无子宫内膜异位症,术后获得输卵管伞及配对子宫内膜。组织标本取得后切成小块液氮速冻或经10%中性福尔马林固定后包埋便于组织学鉴别及研究。包含上皮组织和间质的卵巢子宫内膜异位症病灶组织,输卵管粘膜上皮及相应子宫内膜经显微镜下行微切割,留待后续的实时定量PCR或Western blot蛋白印迹实验。从21例非卵巢子宫内膜异位症患者组织标本中取3对配对的新鲜输卵管伞及子宫内膜组织,进行全基因组表达谱芯片实验并分析找出组织特异性的标记物。基因筛选标准：在两种组织中该基因的表达差异大于2倍且p<0.05。根据芯片结果及筛选标准,选定目的基因：FMO3和DMBTl,并在21例非卵巢子宫内膜异位症患者组织标本中通过实时定量PCR进行进一步验证。利用实时定量PCR分析35例卵巢子宫内膜异位症患者输卵管伞,子宫内膜以及卵巢内异症病灶三处组织中的目的基因表达状况。利用Western Blot蛋白印迹实验在蛋白水平上分析上述组织标本中FM03和DMBTl的表达状况。利用免疫组化技术分析FM03和DMBTl在输卵管伞,子宫内膜和卵巢子宫内膜异位症组织切片中的表达分布状况。
结果：
1.全基因组表达谱芯片分析发现多种差异表达基因在输卵管与子宫内膜中分别有4114个和3451个基因存在差异表达,共有1796个基因表达差异倍数在2倍以上。与子宫内膜比较,输卵管中共有911个上调2倍以上的基因,包括8个50倍以上的基因,28个20倍以上的基因,及875个2倍以上的基因。与输卵管伞比较,子宫内膜中共有885个上调2倍以上的基因,包括7个20倍及以上的基因,878个2倍及以上的基因,尚无上调50倍以上的基因。
2.FM03基因在输卵管伞中高表达,而DMBT1基因在子宫内膜中高表达实时定量PCR实验证实21例非卵巢子宫内膜异位症患者配对的输卵管和子宫内膜组织中2种基因的表达状况：FM03在输卵管中高表达(平均上调倍数44.38,p<0.001),而DMBT1在子宫内膜中高表达(平均上调倍数22.11,p＜0.001)。Western blot蛋白印迹实验显示FM03在输卵管伞中高表达(平均上调倍数11.05,p=0.006),而DMBT1在输卵管伞中低表达(平均下调倍数32.08,p=0.001)。免疫组化显示FM03和DMBT1为细胞浆着色。多数输卵管上皮细胞呈现FM03中等或强阳性染色。21例子宫内膜组织腺体细胞呈弥漫性DMBT1强阳性染色,输卵管上皮细胞尚未见DMBT1阳性染色。
3.FM03及DMBT1在卵巢子宫内膜异位症组织中的表达FM03及DMBT1在35例卵巢子宫内膜异位症患者的输卵管伞和子宫内膜组织中在基因转录水平及蛋白水平的表达趋势与非卵巢子宫内膜异位症患者一致。有32例患者三处组织标本均可用于实时定量PCR分析,与对应的子宫内膜比较,其中18例卵巢子宫内膜异位症组织FM03表达上调,平均上调倍数7.21倍(p=0.016)；与对应的输卵管伞比较,14例卵巢子宫内膜异位症组织标本中FM03表达明显下调,但是与对应的子宫内膜组织比较,没有统计学差异。在18例上调表达FM03的卵巢子宫内膜异位症组织标本中,与对应的子宫内膜组织比较,其DMBT1的表达明显下调,平均下调倍数6.94(p=0.022)；但是与对应输卵管伞比较,没有统计学差异(p=0.144)。Western blot蛋白印迹实验显示上述18例上调表达FM03同时下调表达DMBT1的卵巢子宫内膜异位症组织中FM03及DMBT1蛋白表达趋势同mRNA水平表达趋势,其中FM03蛋白表达平均上调7.2倍。剩余17例卵巢子宫内膜异位症组织标本Western blot蛋白印迹实验分析显示8例与子宫内膜中具有相似的FM03蛋白表达水平,9例与对应子宫内膜中该蛋白表达水平无统计学差异。19例卵巢子宫内膜异位症患者三处组织Western blot蛋白印迹实验显示DMBT1蛋白在卵巢子宫内膜异位症组织中的表达状况与在输卵管伞中表达状况相似,无统计学差异,但子宫内膜组织DMBT1蛋白表达明显上调。剩余16例卵巢子宫内膜异位症患者中,与输卵管伞比较,DMBT1蛋白在卵巢子宫内膜异位症组织中表达上调,其中有12例上调表达显著,另有4例卵巢子宫内膜异位症组织与对应子宫内膜比较没有显示表达差异。35例卵巢子宫内膜异位症组织免疫组化显示,19例子宫内膜异位症上皮细胞的胞浆呈FM03中等阳性染色,剩余16例有10例局灶性阳性着色,6例因上皮组织缺如无法着色。有17例卵巢子宫内膜异位症组织切片呈现DMBT1染色阴性,剩余18例其中10例卵巢子宫内膜异位症阳性染色。
结论：近60%的卵巢子宫内膜异位症病例其子宫内膜异位症病灶组织细胞可能起源于输卵管上皮细胞,40%的卵巢子宫内膜异位症病例的子宫内膜异位症病灶组织细胞可能源自子宫内膜细胞。输卵管上皮细胞是卵巢子宫内膜异位症的一种潜在的组织细胞起源。
Background and Purpose:Endometriosis is defined by the presence of endometrial tissue outside the uterus. It is one of the most common benign gynecologic disorders associated with pelvic pain and infertility. Endometriosis, most commonly involving the ovary, affects approximately10%of women in their reproductive age and up to50%of women suffering infertility and abdominal pain. The patho genesis of endometriosis remains unclear and elusive since it was first described by Von Rokitansky over100years ago. In1920s, Sampson's retrograde menstruation theory has been widely accepted, but remains controversial as retrograde menstruation occurs in up to90%of women in reproductive age but only6%to10%of those women have endometriosis. Retrograde menstruation may explain occurrence of endometriosis within the pelvis or abdominal cavity but fails to explain the presence of endometriosis in remote sites outside the peritoneal cavity. These controversial points led Iwanoff and Meyer to propose the coelomic metaplasia theory, which explained that endometriosis may derive from mesothelial cells through metaplasia. The presence of endometriosis in remote areas and the rare endometriosis in males support the metaplasia theory. Although this metaplastic theory may explain the majority of endometriosis both in and outside of the ovary and within the peritoneal cavity, it lacks a cellular or molecular basis. Lymphatic and vascular transportation of the endometrium has also been proposed as a complimentary theory to explain rare cases of endometriosis occurring in unusual locations far from the pelvis, but this is unlikely to be the primary mechanism of disease spread. Another compelling proposal suggests that bone marrow derived stem cells may differentiate into endometriotic tissue within and outside of endometrial cavity. However, the stem cell theory is premitive and not completely understood. Overall, no single theory perfectly accounts for the pathogenesis of all cases of endometriosis.
The fallopian tube was previously recognized only as a carrier for the menstrual endometrium to pass into the peritoneal cavity or the ovarian surface. The fallopian tube has never been examined as a possible tissue or cellular source of endometriosis. Based on our clinical observations of tubal specimens, we think that the fallopian tube is likely in the certain degree to contribute the formation of ovarian endometriosis. Tubal mucosa is known to be able to form endometrial-like tissue. For instance, endometrialization is commonly seen within the tubal lumen after tubal ligation. It is also known that tubal epithelia shed viable cells onto the ovarian surface forming endosalpingiosis or ovarian epithelial inclusions, a common finding seen within the ovary in approximately30%of the cases. In a recent study onthe cell origin of low-grade serous carcinoma of the ovary, we demonstrated that the majority of the OEIs are derived from tubal epithelia and the OEIs could be transformed into ovarian endometriosis through a probable metaplastic process. We hypothesize that tubal epithelium contributes to the formation of ovarian endometriosis. To examine this provocative hypothesis, we searched for biomarkers which are specific to help differentiate tubal from endometrial origin of endometriosis within the ovary. In this study, we identified a set of novel genes which are either highly expressed in the fallopian tube or in the endometrium through a gene differential array study. We validated these unique genes and their corresponding protein expression in ovarian endometriosis by comparing their expression levels to paired specimens of fallopian tube and endometrium within individual patients.
Material and Methods:Tissue samples including human tubal fimbria, paired endometrium and ovarian endometriosis were obtained from surgical pathology specimens within30minutes of resection at the Department of Gynecology, Qilu Hospital of Shandong University from2010to2013. A total of147specimens derived from56patients were studied. Among them, each of35patients with ovarian endometriosis generated a set of samples including fallopian tube, endometrium, and ovarian endometriosis. The remaining non endometriosis patients generated21paired fallopian tube and endometrial samples. All these patients underwent total hysterectomy and bilateral salpingo-oophorectomy for either ovarian endometriosis or benign gynecologic disease without endometriosis. The patients' age ranged from35to51years with a mean age of41.5. Representative portions of the same tissue specimen were either snap frozen and stored in liquid nitrogen until use or fixed in10%neutral formaldehyde overnight and embedded in paraffin for routine histological examination. Tissue sections containing areas of endometriosis, tubal mucosa and endometrium with both glandular epithelia and stroma were confirmed under microscope and hand microdissected for either real-time PCR or Western analysis. In order to identify tissue specific bio markers, we compared the gene expression between the fallopian tube and the endometrium from patients without evidence of endometriosis by whole-genome microarray analysis. Three pairs of fresh human endometrium and corresponding tubal fimbria specimens were selected from the pool of the21paired samples mentioned above, labeled, and sent to perform whole-genome expression microarray analysis. The criteria for selection of differentially expressed genes were (1) The cut-off value differentially expressed level between the tubal fimbria and the endometrium is more than2-fold;(2) p＜0.05in fold change filtering. Genes that fit these criteria were considered significant for discrimination. The target genes for this study were selected based on the level of expression after comparing with tubal expression:FMO3and DMBT1. To verify the gene expression data obtained from the microarray, real-time PCR was performed on2selected genes, using total RNA from21paired tubal fimbria and corresponding endometrial samples including those three paired specimens for the gene array analysis. Thirty-five cases with ovarian endometriosis and paired tubal and endometrial samples were analyzed by real-time PCR. All samples mentioned above were subsequently evaluated for protein expression of FMO3and DMBT1by Western blot. Immunohistochemistry (IHC) with antibodies to FMO3and DMBT1was performed on the sections of fallopian tubal mucosa, corresponding endometrium and ovarian endometriosis respectively.
Results:
1. Multiple unique genes identified in the fallopian tube over the endometrium A total of4114and3451genes were identified in the fallopian tube and endometrium, respectively. There were1796genes identified with more than2-fold differential expression between human fallopian tube and endometrial tissues. All these differentially expressed genes were further scrutinized and the highly differentially expressed genes were summarized. Compared with the endometrium, the fallopian tube showed a total911up-regulated genes. These included50-fold or more (n=8),20-fold or more (n=28), and2-fold or more (n=875). Compared with the fallopian tube, the endometrium showed a total of885up-regulated genes including20-fold or more (n=7) and2-fold or more (n=878). There were no genes with more than50-fold up-regulation found in the endometrial tissue.
2. FMO3was highly expressed in the fallopian tube, while DMBT1was in the endometrium After screening, we identified2genes, one was highly expressed in the fallopian tube (FM03) and the other in the endometrium (DMBT1), which matched our specified conditions. We validated these two genes with real-time PCR, Western blot and immunohistochemistry. Both the FMO3and DMBT1genes follow the trend differences of the microarray results. In the21pairs of the tubal and corresponding endometrial samples in real-time PCR analysis, FM03was highly expressed in the fallopian tube compared with the paired endometrium, with average fold change of44.38(p＜0.001). In contrast, DMBT1was highly expressed in endometrium compared with the fallopian tube with average fold change of22.11(p＜0.001). None of the21pairs of samples showed discordance with the trend towards co-expression of FMO3and DMBT1in fallopian tube versus endometrial tissue. Among the21pairs of tubal and endometrial samples,14pairs were adequate for Western blot analysis. FMO3protein expression was significantly higher in the fallopian tube samples than that in the endometrium, with an average fold of increment11.05(p=0.006). In contrast, the DMBT1protein level was significantly lower in the fallopian tube (average decreasing fold=32.08) compared with the expression in the endometrium (p＝0.001). These results were compatible with the findings from real-time PCR validation, indicating FMO3and DMBT1do not change significantly at the transcriptional and post-transcriptional levels. All21pairs of the tissue samples were studied for the cellular location of both FM03and DMBT1by immunohistochemistry. Both bio markers were mainly cytoplasmic. No nuclear stainings were identified for these2genes. Of the21paired samples,19(91%) showed moderately to strongly positive staining of FM03in the majority epithelial cells of the fallopian tube, while the remaining2tubal samples were weakly stained. In contrast, FM03was only weakly and focally expressed in the3(14%) endometrial samples, mainly within the endometrial glands. There were some stromal and endothelial cell stainings identified with no specific pattern. DMBT1was strongly and diffusely positive in the majority of glandular cells in all21endometrial samples, but not in the tubal sections we studied.
3. FMO3and DMBT1expression in ovarian endometriosis. There were a total of35patients with the requisite three samples for the study. These gene expression levels were compared in patients with ovarian endometriosis and those without ovarian endometriosis from the data presented above. Both FM03and DMBT1expression levels in the fallopian tubal and endometrial samples showed no statistical difference between the patients with ovarian endometriosis and those without ovarian endometriosis. Among the35cases with ovarian endometriosis,32paired samples were adequate for real-time PCR analysis. FMO3was highly expressed in18(56%) of the32samples studied, with the average fold increment of7.21(p=0.016) compared with the endometrium. However, FMO3expression in the remaining14ovarian endometriosis samples was significantly lower than that in fallopian tube (p<0.01), but similar to the level of expression in the corresponding endometrium (p=0.184). In contrast, the18patients with high level of FM03expression, showed a significantly low expression of DMBT1in the ovarian endometriosis lesions compared with the paired endometrial samples (average decreasing fold=6.94, p=0.022). Meanwhile, DMBT1expression showed no statistical differences between the ovarian endometriosis and fallopian tube samples (p=0.144). The18ovarian endometriosis samples with high FMO3and low DMBT1expression also showed concordant level of the protein expression by Western blot. The FM03expression increased with an average fold increment of7.2(p=0.007) compared with the expression in the endometrium. Among the remaining17ovarian endometriosis samples,8showed a similar level of FMO3expression to the endometrium but lower than the tube, while9showed no statistical difference compared to either the endometrium or the fallopian tube. Among the35paired patients,19cases showed that DMBT1protein expression in ovarian endometriosis samples was similarly low in the fallopian tube, while significantly high in the endometrium. DMBT1in the remaining16cases showed high expression in areas of ovarian endometriosis and the endometrium (n=12) and no difference (n=4) between ovarian endometriosis and paired endometrium. Cellular localization of these gene products was examined by immunohistochemistry. Among the35paired samples,19(54%) showed that FMO3was positively staining in the cytoplasm of ovarian endometriosis epithelium, while the remaining16ovarian endometriosis samples showed either low expression in focal areas (n=10) or inadequate expression for analysis due to loss of glandular epithelia within the sections (n=6). In terms of DMBT1,17(49%) ovarian endometriosis samples were negative, while the remaining18ovarian endometriosis samples showed positive staining (n=10) or were inadequate (n=8).
Conclusion:Based on our preliminary study, the findings suggest that approximately60%of the ovarian endometriosis we studied may be derived from the fallopian tube, while about40%of the cases may be of endometrial origin. The fallopian tube epithelia may represent one of the tissue sources contributing to ovarian endometriosis. Such novel findings, which require confirmation, may have a significant clinical impact in searching for alternative ways of prevention and treatment of endometriosis.
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37. Levander G, Normann P. The pathogenesis of endometriosis; an experimental study. Acta Obstet Gynecol Scand 1955; 34:366-398.
38. Bontis JN, Vavilis DT. Etiopathology of endometriosis. Ann N YAcad Sci 1997; 816:305-309.
39. Vinatier D, Orazi G, Cosson M, et al. Theories of endometriosis. Eur J Obstet Gynecol Reprod Biol 2001; 96:21-34.
40. Matsuura K, Ohtake H, Katabuchi H, et al. Coelomic metaplasia theory of endometriosis:evidence from in vivo studies and an in vitro experimental model. Gynecol Obstet Invest 1999; 47 Suppl 1:18-20; discussion 20-12.
41. Merrill JA. Endometrial induction of endometriosis across Millipore filters. Am J Obstet Gynecol 1966; 94:780-790.
42. Lauchlan SC. The secondary Mullerian system. Obstet Gynecol Surv 1972; 27: 133-146.
43. Sasson IE, Taylor HS. Stem cells and the pathogenesis of endometriosis. Ann N YAcad Sci 2008; 1127:106-115.
44. Taylor HS. Endometrial cells derived from donor stem cells in bone marrow transplant recipients. JAMA 2004; 292:81-85.
45. Du H, Taylor HS. Contribution of bone marrow-derived stem cells to endometrium and endometriosis. Stem Cells 2007; 25:2082-2086.
46. Ikoma T, Kyo S, Maida Y, et al. Bone marrow-derived cells from male donors can compose endometrial glands in female transplant recipients. Am J Obstet Gynecol 2009; 201:608 e601-608.
47. Figueira PG, Abrao MS, Krikun G, et al. Stem cells in endometrium and their role in the pathogenesis of endometriosis. Ann N Y Acad Sci 2011; 1221:10-17.
48. Clement PB. The pathology of endometriosis:a survey of the many faces of a common disease emphasizing diagnostic pitfalls and unusual and newly appreciated aspects. Adv Anat Pathol 2007; 14:241-260.
49. Gardner GHG, R.R. Ranney, B. The histogenesis of endometriosis Recent contributions. Obstet Gynec 1953; 1:615-637.
50. Li J, Abushahin N, Pang S, et al. Tubal origin of 'ovarian' low-grade serous carcinoma. Mod Pathol 2011; 24:1488-1499.
51. Dietl J, Wischhusen J, Hausler SF. The post-reproductive Fallopian tube:better removed? Hum Reprod 2011; 26:2918-2924.
52. Kurman RJ, Shih Ie M. The origin and pathogenesis of epithelial ovarian cancer:a proposed unifying theory. Am J Surg Pathol 2010; 34:433-443.
53. Roh MH, Kindelberger D, Crum CP. Serous tubal intraepithelial carcinoma and the dominant ovarian mass:clues to serous tumor origin? Am J Surg Pathol 2009; 33:376-383.
54. Eddy CA, Pauerstein CJ. Anatomy and physiology of the fallopian tube. Clin Obstet Gynecol 1980; 23:1177-1193.
55. Gordts S, Campo R, Rombauts L, et al. Endoscopic visualization of the process of fimbrial ovum retrieval in the human. Hum Reprod 1998; 13:1425-1428.
56. Piek JM, Kenemans P, Verheijen RH. Intraperitoneal serous adenocarcinoma:a critical appraisal of three hypotheses on its cause. Am J Obstet Gynecol 2004; 191:718-732.
57. Piek JM, van Diest PJ, Zweemer RP, et al. Tubal ligation and risk of ovarian cancer. Lancet 2001; 358:844.
58. Ambekar SV, Mudbhatkal NS, Kothare SN. Post-salpingectomy endometriosis (endosalpingiosis); a report of two cases. J Postgrad Med 1965; 11:141-144.
59. Chakrabarti I, Ghosh N. Post-salpingectomy endometriosis:An under-recognized entity. J Midlife Health 2010; 1:91-92.
60. deHoop TA, Mira J, Thomas MA. Endosalpingiosis and chronic pelvic pain. J Reprod Med 1997; 42:613-616.
61. Nishida M, Watanabe K, Sato N, et al. Malignant transformation of ovarian endometriosis. Gynecol Obstet Invest 2000; 50 Suppl 1:18-25.
62. Casagrande JT, Louie EW, Pike MC, et al.'Incessant ovulation" and ovarian cancer. Lancet 1979; 2:170-173.
63. Chene G, Penault-Llorca F, Le Bouedec G, et al. Ovarian epithelial dysplasia after ovulation induction:time and dose effects. Hum Reprod 2009; 24: 132-138.
64. Fathalla MF. Incessant ovulation--a factor in ovarian neoplasia? Lancet 1971; 2: 163.
65. King SM, Hilliard TS, Wu LY, et al. The impact of ovulation on fallopian tube epithelial cells:evaluating three hypotheses connecting ovulation and serous ovarian cancer. Endocr Relat Cancer 2011; 18:627-642.
66. Ness RB, Cottreau C. Possible role of ovarian epithelial inflammation in ovarian cancer. JNatl Cancer Inst 1999; 91:1459-1467.
67. Risch HA, Howe GR. Pelvic inflammatory disease and the risk of epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 1995; 4:447-451.
68. Ness RB, Modugno F. Endometriosis as a model for inflammation-hormone interactions in ovarian and breast cancers. European Journal of Cancer 2006; 42:691-703.
69. Balkwill F, Mantovani A. Inflammation and cancer:back to Virchow? Lancet 2001; 357:539-545.
70. Chakravarti S, Collins WP, Forecast JD, et al. Hormonal profiles after the menopause. Br Med J 1976; 2:784-787.
71. Vanderhyden BC. Loss of ovarian function and the risk of ovarian cancer. Cell Tissue Res 2005; 322:117-124.
72. Wong AS, Auersperg N. Ovarian surface epithelium:family history and early events in ovarian cancer. Reprod Biol Endocrinol 2003; 1:70.
73. Burmeister RE, Fechner RE, Franklin RR. Endosalpingiosis of the peritoneum. Obstet Gynecol 1969; 34:310-318.
74. Stock RJ. Postsalpingectomy endometriosis:a reassessment. Obstet Gynecol 1982; 60:560-570.
75. Tutschka BG, Lauchlan SC. Endosalpingiosis. Obstet Gynecol 1980; 55: 57S-60S.
76. Resta L, Russo S, Colucci GA, et al. Morphologic precursors of ovarian epithelial tumors. Obstet Gynecol 1993; 82:181-186.
77. Scully RE. Pathology of ovarian cancer precursors. J Cell Biochem Suppl 1995; 23:208-218.
78. Fukunaga M, Ushigome S. Epithelial metaplastic changes in. ovarian endometriosis. Mod Pathol 1998; 11:784-788.
79. Zheng W, Li N, Wang J, et al. Initial endometriosis showing direct morphologic evidence of metaplasia in the pathogenesis of ovarian endometriosis. Int J Gynecol Pathol 2005; 24:164-172.
2. Giudice LC, Kao LC. Endometriosis. The Lancet 2004; 364:1789-1799.
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17. D'Hooghe TM, Debrock S. Endometriosis, retrograde menstruation and peritoneal inflammation in women and in baboons. Hum Reprod Update 2002; 8:84-88.
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19. Chatterjee SK. Scar endometriosis:a clinicopathologic study of 17 cases. Obstet Gynecol 1980; 56:81-84.
20. Suginami H. A reappraisal of the coelomic metaplasia theory by reviewing endometriosis occurring in unusual sites and instances. Am J Obstet Gynecol 1991; 165:214-218.
21. Javert CT. The spread of benign and malignant endometrium in the lymphatic system with a note on coexisting vascular involvement. Am J Obstet Gynecol 1952; 64:780-806.
22. Noel JC, Chapron C, Fayt I, et al. Lymph node involvement and lymphovascular invasion in deep infiltrating rectosigmoid endometriosis. Fertil Steril 2008;89:1069-1072.
23. Reid GD, Kowalski D, Cooper MJ, et al. Hepatic endometriosis:a case report and review of the literature. Aust N Z J Obstet Gynaecol 2003; 43:87-89.
24. Van Schil PE, Vercauteren SR, Vermeire PA, et al. Catamenial pneumothorax caused by thoracic endometriosis. Ann Thorac Surg 1996; 62:585-586.
25. Lucidi RS, Witz CA, Chrisco M, et al. A novel in vitro model of the early endometriotic lesion demonstrates that attachment of endometrial cells to mesothelial cells is dependent on the source of endometrial cells. Fertil Steril 2005; 84:16-21.
26. Hornung D, Ryan IP, Chao VA, et al. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol Metab 1997; 82:1621-1628.
27. Kao LC, Germeyer A, Tulac S, et al. Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation Mure and infertility. Endocrinology 2003; 144:2870-2881.
28. Osteen KG, Bruner KL, Sharpe-Timms KL. Steroid and growth factor regulation of matrix metalloproteinase expression and endometriosis. Semin Reprod Endocrinol 1996; 14:247-255.
29. Tseng JF, Ryan IP, Milam TD, et al. Interleukin-6 secretion in vitro is up-regulated in ectopic and eutopic endometrial stromal cells from women with endometriosis. J Clin Endocrinol Metab 1996; 81:1118-1122.
30. Wu Y, Kajdacsy-Balla A, Strawn E, et al. Transcriptional characterizations of differences between eutopic and ectopic endometrium. Endocrinology 2006; 147:232-246.
31. Nair AS, Nair HB, Lucidi RS, et al. Modeling the early endometriotic lesion: mesothelium-endometrial cell co-culture increases endometrial invasion and alters mesothelial and endometrial gene transcription. Fertil Steril2008; 90: 1487-1495.
32. Dmowski WP, Gebel HM, Rawlins RG. Immuno logic aspects of endometriosis. Obstet Gynecol Clin North Am 1989; 16:93-103.
33. Osteen KG, Sierra-Rivera E. Does disruption of immune and endocrine systems by environmental toxins contribute to development of endometriosis? Semin Reprod Endocrinol 1997; 15:301-308.
34. Schrodt GR, Alcorn MO, Ibanez J. Endometriosis of the male urinary system:a case report. J Urol 1980; 124:722-723.
35. Batt RE, Smith RA. Embryologic theory of histogenesis of endometriosis in peritoneal pockets. Obstet Gynecol Clin North Am 1989; 16:15-28.
36. Olive DL, Franklin RR, Gratkins LV. The association between endometriosis and spontaneous abortion. A retrospective clinical study. JReprod Med 1982; 27:333-338.
37. Levander G, Normann P. The pathogenesis of endometriosis; an experimental study. Acta Obstet Gynecol Scand 1955; 34:366-398.
38. Bontis JN, Vavilis DT. Etiopathology of endometriosis. Ann N YAcad Sci 1997; 816:305-309.
39. Vinatier D, Orazi G, Cosson M, et al. Theories of endometriosis. Eur J Obstet Gynecol Reprod Biol 2001; 96:21-34.
40. Matsuura K, Ohtake H, Katabuchi H, et al. Coelomic metaplasia theory of endometriosis:evidence from in vivo studies and an in vitro experimental model. Gynecol Obstet Invest 1999; 47 Suppl 1:18-20; discussion 20-12.
41. Merrill JA. Endometrial induction of endometriosis across Millipore filters. Am J Obstet Gynecol 1966; 94:780-790.
42. Lauchlan SC. The secondary Mullerian system. Obstet Gynecol Surv 1972; 27: 133-146.
43. Sasson IE, Taylor HS. Stem cells and the pathogenesis of endometriosis. Ann N YAcad Sci 2008; 1127:106-115.
44. Taylor HS. Endometrial cells derived from donor stem cells in bone marrow transplant recipients. JAMA 2004; 292:81-85.
45. Du H, Taylor HS. Contribution of bone marrow-derived stem cells to endometrium and endometriosis. Stem Cells 2007; 25:2082-2086.
46. Ikoma T, Kyo S, Maida Y, et al. Bone marrow-derived cells from male donors can compose endometrial glands in female transplant recipients. Am J Obstet Gynecol 2009; 201:608 e601-608.
47. Figueira PG, Abrao MS, Krikun G, et al. Stem cells in endometrium and their role in the pathogenesis of endometriosis. Ann N Y Acad Sci 2011; 1221:10-17.
48. Clement PB. The pathology of endometriosis:a survey of the many faces of a common disease emphasizing diagnostic pitfalls and unusual and newly appreciated aspects. Adv Anat Pathol 2007; 14:241-260.
49. Gardner GHG, R.R. Ranney, B. The histogenesis of endometriosis Recent contributions. Obstet Gynec 1953; 1:615-637.
50. Li J, Abushahin N, Pang S, et al. Tubal origin of 'ovarian' low-grade serous carcinoma. Mod Pathol 2011; 24:1488-1499.
51. Dietl J, Wischhusen J, Hausler SF. The post-reproductive Fallopian tube:better removed? Hum Reprod 2011; 26:2918-2924.
52. Kurman RJ, Shih Ie M. The origin and pathogenesis of epithelial ovarian cancer:a proposed unifying theory. Am J Surg Pathol 2010; 34:433-443.
53. Roh MH, Kindelberger D, Crum CP. Serous tubal intraepithelial carcinoma and the dominant ovarian mass:clues to serous tumor origin? Am J Surg Pathol 2009; 33:376-383.
54. Eddy CA, Pauerstein CJ. Anatomy and physiology of the fallopian tube. Clin Obstet Gynecol 1980; 23:1177-1193.
55. Gordts S, Campo R, Rombauts L, et al. Endoscopic visualization of the process of fimbrial ovum retrieval in the human. Hum Reprod 1998; 13:1425-1428.
56. Piek JM, Kenemans P, Verheijen RH. Intraperitoneal serous adenocarcinoma:a critical appraisal of three hypotheses on its cause. Am J Obstet Gynecol 2004; 191:718-732.
57. Piek JM, van Diest PJ, Zweemer RP, et al. Tubal ligation and risk of ovarian cancer. Lancet 2001; 358:844.
58. Ambekar SV, Mudbhatkal NS, Kothare SN. Post-salpingectomy endometriosis (endosalpingiosis); a report of two cases. J Postgrad Med 1965; 11:141-144.
59. Chakrabarti I, Ghosh N. Post-salpingectomy endometriosis:An under-recognized entity. J Midlife Health 2010; 1:91-92.
60. deHoop TA, Mira J, Thomas MA. Endosalpingiosis and chronic pelvic pain. J Reprod Med 1997; 42:613-616.
61. Nishida M, Watanabe K, Sato N, et al. Malignant transformation of ovarian endometriosis. Gynecol Obstet Invest 2000; 50 Suppl 1:18-25.
62. Casagrande JT, Louie EW, Pike MC, et al.'Incessant ovulation" and ovarian cancer. Lancet 1979; 2:170-173.
63. Chene G, Penault-Llorca F, Le Bouedec G, et al. Ovarian epithelial dysplasia after ovulation induction:time and dose effects. Hum Reprod 2009; 24: 132-138.
64. Fathalla MF. Incessant ovulation--a factor in ovarian neoplasia? Lancet 1971; 2: 163.
65. King SM, Hilliard TS, Wu LY, et al. The impact of ovulation on fallopian tube epithelial cells:evaluating three hypotheses connecting ovulation and serous ovarian cancer. Endocr Relat Cancer 2011; 18:627-642.
66. Ness RB, Cottreau C. Possible role of ovarian epithelial inflammation in ovarian cancer. JNatl Cancer Inst 1999; 91:1459-1467.
67. Risch HA, Howe GR. Pelvic inflammatory disease and the risk of epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 1995; 4:447-451.
68. Ness RB, Modugno F. Endometriosis as a model for inflammation-hormone interactions in ovarian and breast cancers. European Journal of Cancer 2006; 42:691-703.
69. Balkwill F, Mantovani A. Inflammation and cancer:back to Virchow? Lancet 2001; 357:539-545.
70. Chakravarti S, Collins WP, Forecast JD, et al. Hormonal profiles after the menopause. Br Med J 1976; 2:784-787.
71. Vanderhyden BC. Loss of ovarian function and the risk of ovarian cancer. Cell Tissue Res 2005; 322:117-124.
72. Wong AS, Auersperg N. Ovarian surface epithelium:family history and early events in ovarian cancer. Reprod Biol Endocrinol 2003; 1:70.
73. Burmeister RE, Fechner RE, Franklin RR. Endosalpingiosis of the peritoneum. Obstet Gynecol 1969; 34:310-318.
74. Stock RJ. Postsalpingectomy endometriosis:a reassessment. Obstet Gynecol 1982; 60:560-570.
75. Tutschka BG, Lauchlan SC. Endosalpingiosis. Obstet Gynecol 1980; 55: 57S-60S.
76. Resta L, Russo S, Colucci GA, et al. Morphologic precursors of ovarian epithelial tumors. Obstet Gynecol 1993; 82:181-186.
77. Scully RE. Pathology of ovarian cancer precursors. J Cell Biochem Suppl 1995; 23:208-218.
78. Fukunaga M, Ushigome S. Epithelial metaplastic changes in. ovarian endometriosis. Mod Pathol 1998; 11:784-788.
79. Zheng W, Li N, Wang J, et al. Initial endometriosis showing direct morphologic evidence of metaplasia in the pathogenesis of ovarian endometriosis. Int J Gynecol Pathol 2005; 24:164-172.