The Expression And Significance Of Midkine(MK), Pleiotrophin(PTN) And Hepatocyte Growth Factor (HGF/SF) In Endometriosis

Objective: Endometriosis(EMs)is a common gynecologic- al disease of reproductive-aged women.Although Ems is a benigh disease,there are many biological characteristics such as implantation, infiltration and metastasis-like malignant tumor. The cause of EMs remains obscure. Sampson, in his theory of retrograde menstruation that had won general recognition, believed that the endometrial cells and fragments sheded during the menstrual period and were transported through the fallopian tubes into the peritoneal cavity where they implanted, proliferated and developed into endometriotic lesions.However, several scientists believe that nearly all the women have the phenomena of retrograde menstruation,but it is by no means all the women suffer from endometriosis.It can be presumed that there are, inevitably,some driving factors presented in the processes of endometriosis caused by retrograde menstruation. Recently, lots of investigators believe that angiogenesis is a crucial phenomenon in the development of endometriosis. Midkine family is a new kind of heparin-binding growth factor which contains two members-midkine(MK)and pleiotrophin (PTN). Midkine family is one of the important factors promoting angiogenesis,stimulating the proliferation of vascular endothelial cell,and can apparently regulate new angiogenesis. Hepatocyte growth factor (HGF) is also called scatter factor(SF). Scientists have proved that HGF/SF produced by the mesenchymal cells of mankind and gnawer and can promote cell proliferation, migration, morphogenic and angiogenesiscanal binding with its receptor c-met. In the study,we investigate the expression and clinical significance of midkine(MK),pleiotrophin(PTN) and HGF/SF in endometriosis. And thus we explored the cause mechanism of endometriosis further.Methods: Group of patients with endometriosis: A total of 35 women between 22 and 50 years old undergoing laparotomy or laparoscopy for endometriosis in the Department of Obstetrics and Gynecology of the Second Affiliated Hospital in the Medical University of Hebei. Diagnosis of endometriosis was established laparoscopically and histologically. According to revised American Fertility Society Classification(1985),10 of the 35 endometriosis patients were classified in stage I and II,and 25 in stage III and IV. All the women had normal menstrual cycles. The phase of menstrual cycle of the women was determined by their last menstrual period. 35 were from women with endometriosis( follicular phase n =19, luteal phase n=16).Take their ectopic and eutopic endometria during laparoscopy or laparotomy .Take the peritoneal fluid of 20 women with endometriosis ( follicular phase n =10, luteal phase n=10) during laparoscopy or laparotomy. Collect their peripheral blood before operations. Control group: 25 women were from non-endometriosis patients who were such conditions as infertility or ovarian dysembryoma or simple cyst(follicular phase n =12, luteal phase n =13). Take their ectopic and eutopic endometria during laparoscpy or lapartomy . Take the peritoneal fluid of 15 women from non-endometriosis patients ( follicular phase n =6, luteal phase n=9)during laparoscopy or laparotomy. Collect their peripheral blood before operations. Immediately after collection, the endometria were washed with normal saline and were put into liquid nitrogen quickly.The peritoneal fluid and peripheral blood samples were centrifuged at 3000 rpm for 10 minutes at 4℃,and the supernatants were aliquoted and stored at-80℃until analyzed. In the study,we used RT-PCR method to investigate the expression and clinical significance of midkine(MK),pleiotrophin(PTN) and HGF/SF in the ectopic and eutopic endometria of endometriosis patients and endometria of normal women.In addition,we used ELISA method to test the expression and significance of HGF/SF in the peritoneal fluid and peripheral blood samples of endometriosis patients and normal women.The statistical software SPSS14.0 was used to analyze the data.Results:1 In the normal endometria of non-endometriosis patients, the eutopic and ectopic endometria with endometriosis , MK,PTN and HGF/SF mRNA were detected by RT-PCR:⑴In the normal endometria of non-endometriosis patients, the eutopic and ectopic endometria of patients with endometriosis , the expression of MK was 0.494±0.191,0.848±0.209,0.542±0.159 respectively. The expression of MK in the the eutopic endometrium with endometriosis was higher than that in the ectopic endometrium with endometriosis and in the normal endometrium of non-endometriosis patients.The differences were markedly significant(all p<0.05). The expression of MK in ectopic endometrium of patients with endometriosis was little higher than that in endometrium of the normal women . But the difference was not markedly significant(p =0.32 and p >0.05).⑵In the normal endometrium of non-endometriosis patients, the eutopic and ectopic endometrium with endometriosis , the expression of PTN was 0.301±0.096,0.697±0.347,0.368±0.094 respectively. The expression of PTN in the the eutopic endometrium with endometriosis was higher than that in the ectopic endometrium with endometriosis and in the normal endometrium of non-endometriosis patients.The differences were markedly significant(all p<0.05). The expression of pleiotrophin in ectopic endometrium of patients with endometriosis was higher than that in endometrium of the normal women . The difference was also markedly significant(p <0.05).⑶The expression of HGF/SF mRNA in eutopic and ectopic endometrium of patients with endometriosis(0.820±0.232, 0.908±0.361)was higher than that of non-endometriosis women(0.461±0.108).And all p<0.01.There is no marked difference of the expression of HGF/SF mRNA between eutopic and ectopic endometrium of patients with endometriosis(p >0.05)。2 The expression of MK and PTN in eutopic endometria in different stages: The expression of MK and PTN in the eutopic endometria of patients with endometriosis was directly correlated with the clinical stages of the endometriosis.⑴The expression of MK in the eutopic endometria of patients with endometriosis inⅠ,Ⅱstage was0.665±0.134,and that of patients with endometriosis inⅢ,Ⅳstage was 0.922±0.189 (correlation coefficientγ=0.595, p=0.000).⑵The expression of PTN in the eutopic endometria of patients with endometriosis inⅠ,Ⅱstage was 0.450±0.208,and that of patients with endometriosis inⅢ,Ⅳstage was 0.795±0.344(correlation coefficientγ=0.488, p=0.003).⑶The expression of HGF/SF in the eutopic endometria of patients with endometriosis inⅠ,Ⅱstage was 0.590±0.110,and that of patients with endometriosis inⅢ,Ⅳstage was1.036±0.349 (correlation coefficientγ=0.683, p=0.000<0.05).3 The expression of MK and PTN in endometriosis with different period of menstrual cycle:⑴The expression of MK mRNA of endometria of patients with endometriosis in follicular phase and luteal phase are 0.835±0.228,0.864±0.190 respectively. But the difference was not markedly significant(p=0.68 and p>0.05).In the control group, the expression of MKmRNA of endometria in follicular phase and luteal phase are 0.584±0.187,0.411±0.159 respectively. The expression of MKmRNA of endometria in follicular phase are higher than that in luteal phase.And the difference was markedly significant (p=0.021and p<0.05). Neither in follicular phase nor in luteal phase ,the expression of MKmRNA of eutopic endometria of patients with endometriosis was higher than endometria of non-endometriosis(p<0.05 ).⑵The expression of PTNmRNA of endometria of patients with endometriosis in follicular phase and luteal phase are 0.663±0.250,0.736±0.441 respectively. But the difference was not markedly significant(p=0.54 and p>0.05) .In the control group, the expression of PTNmRNA of endometria in follicular phase and luteal phase are 0.290±0.089, 0.310±0.104 respectively. But the difference was not markedly significant(p=0.60 and p>0.05). Neither in follicular phase nor in luteal phase ,the expression of PTNmRNA of eutopic endometria of patients with endometriosis was higher than endometria of non-endometriosis (p<0.05 ).⑶The expression of HGF/SF mRNA of endometria of patients with endometriosis in luteal phase( 1.064±0.381) was higher than that in follicular phase(0.778±0.294).And p=0.021<0.05. The expression of HGF/SF mRNA of endometria of non-endometriosis women in luteal phase(0.502±0.086) was higher than that in follicular phase(0.415±0.115).And p=0.043, p<0.05 Neither in follicular phase nor in luteal phase ,the expression of HGF/SF mRNA of eutopic endometria of patients with endometriosis were higher than endometria of non- endometriosis(all p<0.05 ).4 The expression of HGF/SF in PF and serum:⑴In PF: The expression of HGF/SF in PF of endometriosis patients(1.546±0.214ng/ml) is higher than that of non-endometriosis women (1.124±0.128 ng/ml).And p<0.01. The expression of HGF/SF in PF of endometriosis patients inⅢ,Ⅳstage( 1.614±0.224 ng/ml) is higher than that of endometriosis patients inⅠ,Ⅱstage(1.355±0.239) and non-endometriosis women.The differences were markedly significant( p<0.05 and p<0.01). The expression of HGF/SF in PF of endometriosis patients was directly correlated with the clinical stages of the endometriosis(correlation coefficientγ=0.627, p=0.003<0.01). The expression of HGF/SF in PF of endometriosis patients in follicular phase and luteal phase are 1.479±0.192 ng/ml,1.614±0.224 ng/ml respectively.But p=0.166>0.05, the difference was not markedly significant. The expression of HGF/SF in PF of non-endometriosis women in follicular phase and luteal phase are 1.085±0.100 ng/ml,1.151±0.144 ng/ml respectively.And p=0.313>0.05, the difference was not markedly significant. Neither in follicular phase nor in luteal phase ,the expression of HGF/SF in PF of patients with endometriosis were higher than that of non- endometriosis in corresponding stage(all p<0.05 ).⑵In serum:The expression of HGF/SF in peripheral blood serum of endometriosis patient(s0.523±0.127 ng/ml) is higher than that of non-endometriosis women ( 0.258±0.065 ng/ml).And p<0.01. The expression of HGF/SF in PF of endometriosis patients inⅢ,Ⅳstage(0.573±0.111ng/ml) is higher than that of endometriosis patients inⅠ,Ⅱstage(0.431±0.104 ng/ml) and non-endometriosis women.The differences were markedly significant ( all p<0.01). The expression of HGF/SF in serum of endometriosis patients was directly correlated with the clinical stages of the endometriosis (correlation coefficientγ=0.518,p=0.019<0.05). The expression of HGF/SF in serum of endometriosis patients in luteal phase(0.583±0.120 ng/ml)is higher than that in follicular phase( 0.464±0.108 ng/ml ) . The difference was markedly significant( p<0.05). The expression of HGF/SF in serum of non-endometriosis patients in luteal phas(e0.285±0.054 ng/ml)is higher than that in follicular phase(0.217±0.060 ng/ml). The difference was markedly significant( p<0.05). Neither in follicular phase nor in luteal phase ,the expression of HGF/SF in serum of patients with endometriosis were higher than that of non-endometriosis in corresponding stage(all p<0.05 ).Conclusion: 1 The expression of MK, PTN and HGF/SF in the the eutopic endometrium with endometriosis was higher than that in the normal endometrium of non-endometriosis.All of these can support the theory of"eutopic endometrium determinism". 2 The expression of MK, PTN and HGF/SF in the eutopic endometrium of patients with endometriosis were directly correlated with the clinical stages of the endometriosis. 3 Neither in follicular phase nor in luteal phase ,the expression of MK, PTN and HGF/SF mRNA of eutopic endometria of patients with endometriosis were higher than endometria of non-endometriosis(all p<0.05 ).These can reveal that the strong ability of angiogenesis of patients with endometriosis run though the whole menstrual cycle ,and thus result in the disease persistent. 4 The expression of HGF/SF in PF and serum of patients with endometriosis was higher than that of non-endometriosis women.And it can suggest that the changes of the abdominal cavity and peripheral circulation environment are important for the formation of endometriosis. 5 The expression of HGF/SF in the PF and serum of peripheral blood of patients with endometriosis were directly correlated with the clinical stages of the endometriosis.So examine the expression of HGF/SF in the PF and serum may help the doctors diagnose the disease-endometriosis and evaluate the prognosis.6 The angiogenesis factors-MK, PTN and HGF/SF may play an important role in the formation of endometriosis.It will be very significant to explore the potency of midkine ,pleiotrophin and HGF/SF inhibitor to cure endometriosis.