Detection Of Discrepant Mitochondrial Proteins In Eutopic Endometrium Of Endometriosis And Adenomyosis Using SELDI-TOF-MS

Endometriosis is a chronic condition characterized by the presence of endometnal tissue outside of the uterus in lesions of varying sizes and appearance containing endometrial glands and stroma. The pathogenesis of this enigmatic disorder still remains controversial despite multiple theories regarding the etiology exist. Among them, the implantation hypothesis of retrograde menstruation suggested by Sampson is the most widely accepted one. However, the implantation theory doesn't explain that adhesion and persistence of endometriotic lesions in the pelvis only happen in approximately 10%-15% of women in reproductive age while the retrogradation of endometrial tissues via fallopian tubes during menstruation happens in approximately 80%-90% menstrual cycles. It is assumed that additional factors favor the adhesion and growth of endometrial cells that spill into the peritoneal cavity. Some studies indicate that eutopic endometrium of women affected by endometriosis is abnormal compared with that of healthy women. Although the morphology of eutopic endometrium from women with endometriosis is similar to the normal, its physiology and biochemistry are different.Adenomyosis is characterized by the presence of endometrial glands and stroma within the myometrium. The cause(s) and mechanisms of development of intramyometrial endometrium are not clear. At present, three theories have been proposed, but unanimous agreement has yet to be reached. The most popular hypothesis is that adenomyosis originates from the deep part (basalis) of the endometrial mucosa. The latter would invaginate between bundles of smooth muscle fibres of the myometrium, possibly due to loss of tissue cohesion caused by specific enzymes. About 30% of patients with endometriosis also have adenomyosis, while about 15% of patients with adenomyosis also have endometriosis. Some believed that adenomyosis is generally considered a variant of endometriosis widely referred to as internal endometriosis. While some believed that adenomyosis is a distinct disease from endometriosis requiring different therapeutic approaches.Despite of significant improvement of the diseases the pathogenesis of endometriosis and adenomyosis is still unclear. Opration approaches remain the gold standard for the diagnosis of endometriosis and adenomyosis. It has inherent limitations, and the patients suffer from the symptoms and complications a lot. Therefor it is urgent to get a better understanding of disease and identify groups of proteins involved in its pathogenesis and find sensitive, specific and convenient new biomarkers.To the best of our knowledge, the cellular function of mitochondria, however, is not limited to bioenergetics. They play crucial roles in the metabolism of amino acids and lipids, the biosynthesis of heme and iron-sulfur clusters, cell signaling, and apoptosis. Mitochondria might be new markers for the early detection, risk assessment, and diagnosis of cancer and other diseases, and for the identification of new targets for therapeutic prevention and intervention. Few reports were about the dysfunctions of mitochondria in eutopic endometria of endometriosis and adenomyosis. It was reported that oxidative stress and mitochondrial DNA mutations might be anticipated in the initiation or progression of endometriosis.Protein but not nuclear acid is the material executant and embodiment of life. So the studies on proteomics are preferred to approach to the pathogenesis of endometriosis and adenomyosis and screen sensitive and specific biomarkers. A novel technology, surface-enhanced laser desorption/ionization time-off light mass spectrometry (SELDI-TOF-MS) carved out such a new path to proteomics research on endometriosis and adenomyosis. It can directly obtain high-throughput protein profilings from clinical samples with high sensitive and this is the main advantage of this technology. The present study focused on the discrepant mitochondrial proteins of eutopic endometria from patients with endometriosis, adenomyosis and controls, using SELDI-TOF-MS and bioinformatics tools; establishing diagnostic models of endometriosis and adenomyosis; comparing the discrepant mitochondrial proteins screened with those in serum and eutopic endometrial. The purposes of the study are to screen novel biomarkers of endometriosis and adenomyosis that are fit for diagnosis and monitoring of endometriosis and adenomyosis, to provide materials and elements for the further studies such as the separation, purification and decoding of biomarkers and to provide new ideas of pathogenesis and therapeutic approaches of endometriosis and adenomyosis.Part one Detection of discrepant mitochondrial proteins in eutopic endometrium of endometriosis using SELDI-TOF-MSObjective:To detect specific mitochondrial proteins in eutopic endometrial samples from women with and without endometriosis and build diagnostic models.Methods:Eutopic endometrial samples from women with endometriosis (excluding adenomyosis) (n=24) and women with benign indications as control (n=29) were studied, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein chip technology. After finding the biomarkers, the diagnostic model was evaluated and validated by leave-one cross validation.χ2 test was used to evaluate the sensitivity and specificity of different diagnostic models. T-test was used to evaluate the protein peaks in different rAFS stages of endometriosis.Results:1.78 qualified mitochondrial protein peaks were detected in eutopic endometrium from women with and without endometriosis and 10 of them had a significant difference (P＜0.05). The 5,574 m/z and 9,378 m/z peaks were expressed at a lower level in endometriosis than in controls; the other 8 peaks were expressed in the opposite way.5,574,7,573 and 7,965m/z peaks were from proliferation phase and the rest 7 peaks were from secretory phase.2. Three combined potential biomarkers, with m/z of 15,334,15,128 and 16,069, were found and the diagnostic system (pattern 1) distinguished endometriosis from control samples with a specificity of 86.2% and a sensitivity of 87.5%.3.69 qualified mitochondrial protein peaks were detected in eutopic endometrium from women with endometriosis and controls in their secretory phase and 14 of them had a significant difference (P＜0.05). The 5,367,5,432,9,526 and 9,378 m/z peaks were expressed at a lower level in endometriosis than in controls; the other 10 peaks were expressed in the opposite way.4. Three combined potential biomarkers in secretory phase, with m/z of 7,615, 5,432 and 15,867, were found and the diagnostic system (pattern 2) distinguished endometriosis from control samples with a specificity of 76.9% and a sensitivity of 80.0%. The specificity and sensitivity had no significant difference from those of pattern 1.5.64 qualified mitochondrial protein peaks were detected in eutopic endometrium from women with endometriosis and controls in their proliferation phase and none of them have a significant difference (P＞0.05).6. Two combined potential biomarkers in secretory phase, with m/z of 3,456 and 15,128, were found and the diagnostic system (pattern 3) distinguished endometriosis from control samples with a specificity of 87.5% and a sensitivity of 85.7%.7. Among the 10 discrepant mitochondrial protein peaks in eutopic endometrium from women with and without endometriosis which had a significant difference (P＜0.05), peaks with m/z of 15,334,15,128,7,573,15,868 and 7,982 were expressed at a higher level in endometriosis of stageⅢ&Ⅳthan of stageⅠ&Ⅱ.5,574 and 9,378 m/z peaks were expressed at an opposite way.Part two Detection of discrepant mitochondrial proteins in eutopic endometrium of adenomyosis using SELDI-TOF-MSObjective:To detect specific mitochondrial proteins in eutopic endometrial samples from women with and without adenomyosis and build diagnostic models, and comparie the discrepant mitochondrial proteins screened with those in part one.Methods:Eutopic endometrial samples from women with adenomyosis (n=13) and women with benign indications as control (n=29) were studied, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein chip technology. After finding the biomarkers, the diagnostic model was evaluated and validated by leave-one cross validation.Results:1.82 qualified mitochondrial protein peaks were detected in eutopic endometrium from women with and without adenomyosis and 1 of them had a significant difference (P＜0.05).2. Three combined potential biomarkers, with m/z of 7,614,14,978 and 6,370, were found and the diagnostic systemdistinguished adenomyosis from control samples with a specificity of 93.1% and a sensitivity of 84.6%.3.14 eutopic mitochondrial protein peaks were detected in both endometriosis and adenomyosis patients. The 3,499m/z peak had a significant difference (P=0.029).3,384, 3,499,4,237 and 5,388m/z peaks had the same trend in the two groups.Conclusions:1. Discrepant mitochondrial proteins of eutopic endometria from patients with endometriosis and adenomyosis were detected, which might play a role in the pathogenesis of the two diseases.2. Most of the discrepant eutopic mitochondrial proteins were found in the secretory phase. It was indicated that mitochondrial proteins at secretory phase had a high relationship to the characteristics of endometriosis.3. There were 7 peaks which had a correlation with the rAFS stages of endometriosis. They might play a role in the development of endometriosis.4. There were no similar peaks detected in eutopic mitochondrial proteins and serum from women with endometriosis. Mass/Charge of the proteins might change after being modified and sheared from cells to the circulation system.5. Three eutopic peaks were detected in both mitochondrial proteins and total proteins from women with endometriosis. Purification and identificatoin of the proteins will be an important part of our future study.6. The model composed by 15,334,15,128 and 16,069 m/z could do the best in division of endometriosis and controls. 7. The model composed by 7,614,14,978 and 6,370 m/z could do the best in division of adenomyosis and controls.8. 14 eutopic mitochondrial protein peaks were detected in both endometriosis and adenomyosis patients. Mitochondrial functions might have similar changes in the two diseases, but not totally the same.