| Recurrent abortion is a frequently occurring and difficult to treat disease in women of reproductive age, whose etiology is complicated. Due to recent increase of attention by researchers at home and abroad regarding the prethrombotic state (PTS), which is the persistence of high blood coagulation state, caused by thrombus formation tendency, it is thus believed to be one of the important causes behind fetal deaths and recurrent spontaneous miscarriage. At present, Western practitioners mainly prescribe aspirin and low molecular weight heparin anticoagulant therapy to treat prethrombotic state of recurrent spontaneous miscarriage. But due to the presence of hemorrhage, gastrointestinal discomfort and other adverse reactions, the clinical appliance has been limited. In recent years, the antithrombotic effect of Chinese herbs has been confirmed, showing unique advantages in the treatment of recurrent miscarriage. Thus, the treatment of prethrombotic state of recurrent spontaneous miscarriage, using Chinese Herbs, has become a hot research topic. Our previous research in this area covering 10 years with about 2000 cases of clinical observation, has shown that using Chinese herbs to tonify kidneys, invigorate and nourish blood has been very effective. But a more systematic and in-depth research has not been carried out yet. Chinese medicine, which advocates "nourishing what could be harmed", can play a role of "preventing possible complications before the pregnancy" in treatment of prethrombotic state of recurrent spontaneous miscarriage. Clinical group’s early stage of treatment, using Chinese herbs to tonify kidneys, invigorate and nourish blood, achieved significant clinical curative effect, and less adverse reactions. Does that mean that kidney deficiency and blood stasis is the main syndrome for prethrombotic state of recurrent spontaneous miscarriage? If it is so, should that differ from non-"kidney deficiency and blood stasis" syndrome in endometrial histopathology? And what differences should there be with proteomics? In order to answer these scientific questions, we have carried out this study, starting from TCM syndrome analysis, moving to histopathology verification and also looking into microscopic proteome. We hope to add in-depth analysis on treatment of prethrombotic state of recurrent spontaneous miscarriage, and provide valuable help for prevention and treatment of this disease.Object1 By means of study on the TCM syndrome manifestations of prethrombotic recurrent spontaneous abortion, to explore its main syndrome manifestations.2 By means of study on the differences in endometrial receptivity and histopathology of various TCM syndrome manifestations of prethrombotic recurrent spontaneous abortion, to further analyze the correlation between the kidney deficiency and blood stasis syndrome and the prethrombotic recurrent spontaneous abortion.3 By means of study on the proteomic differences of kidney deficiency and blood stasis syndrome and non kidney deficiency and blood stasis syndrome in the manifestations of prethrombotic recurrent spontaneous abortion, with protein-chip technique, to explore the different protein expressions of various syndrome manifestations.Methods1 According to "Chinese Gynecology", and "Guidelines for Common Gynecology Common Disease in Chinese Medicine" as well as other related literatures, a database of elaborately designed tables was established to facilitate the research. With strict quality control,205 cases of prethrombotic recurrent spontaneous abortion patients were input to the database. The characteristics and distribution thereof were explored, by adopting frequency statistics, cluster analysis and logistic regression methods based on SAS software.2 Based on the TCM syndrome study of the first part after clustered, the correlation between kidney deficiency and blood stasis in endometrial tissues, and the prethrombotic recurrent spontaneous abortion is further analyzed.60 patients were chosen, with 15 suffering blood stasis and kidney deficiency,15 suffering spleen-kidney deficiency,15 suffering Qi-blood deficiency and 15 suffering kidney essence deficiency. At luteal phase, HE dyed endometrial tissues were collected using one-time endometrial tube to observe the endometrial glands development as well as to observe Pinopode distribution under TEM. During ovulation, Endometrial blood flow perfusion were inspected using ultrasonic, which includes the endometrial thickness, morphology, blood type, endometrial blood flow pulsatility index (PI), endometrial blood flow resistance index (RI). At luteal phase, the prethrombotic-stated related platelet-derived growth factor family including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and fibrinolytic system (t-PA, PAI-1) was inspected using ELLSA method. A correlation analysis was done to the indicators including endometrial thickness, endometrium, endometrial blood flow, PI, RI, PAI-1, t-PA, VEGF, PDGF-AA correlation analysis, of the 60 patients thereof, from the perspective of histopathology, ultrasonic, and molecule biology. And the differences of the indicators in different syndromes were further discussed.3 Based on the findings in the first two parts, we continue to do protein expression spectrum analysis to the serum samples using protein-chip technique, to understand the difference in protein expression of various syndrome manifestations. After targeting the different protein and customizing the protein chip accordingly, we would expand the sample quantity for verification.1. Extract 6 serum samples from women with normal pregnancy and childbearing history (normal),12 serum samples from the prethrombotic recurrent abortion patients with kidney deficiency and blood stasis syndrome, and 11 serum samples from the prethrombotic recurrent abortion patients with non kidney deficiency and blood stasis syndrome.2. Extract the protein expression spectrum of samples, using Raybiotech protein chip (item: AAH-BLG-1000 index number:1000), and analyze the protein of difference from sample groups, which is presented in main component charts.3. Use the cluster map and the Venn diagram to select the differently-expressed protein, and do significance analysis and KEGG pathway/pathway-net analysis to the selected protein, after which the prethrombotic recurrent spontaneous abortion related protein is obtained.4. Customize the chip in accordance with the selected key protein, and add 30 kidney deficiency and blood stasis samples as well as 30 non kidney deficiency and blood stasis samples for verification.Results1 Recurrent miscarriage symptoms prothrombotic state in patients with 205 cases and the frequency distribution of cluster analysis, including 87 cases with kidney and blood stasis syndrome most performance, followed by both spleen and kidney, qi syndrome, kidney essence loss deficiency; after frequency statistics and cluster analysis, the kidney and blood stasis syndrome of recurrent miscarriage thrombotic state before the main card, the proportion was 47.28%, higher frequency of symptoms:menstrual color dark red, weak limp, dull, chest tingling, tinnitus, back pain, dark purple tongue with petechiae, pulse astringent.2 ①different syndromes before thrombosis state of patients with recurrent spontaneous abortion on endometrial receptivity morphological features:HE staining,60 cases in the sample,45 cases of endometrial endometrial better type, wherein Kidney Blood 8 patients, accounting for 17.78% less than the other three groups of syndromes; another 15 cases of endometrial worse type endometrial, kidney and blood stasis in which 7 cases, accounting for 46.67%, more than the other three groups syndromes. Chi-square test four groups difference between groups was not significant (P= 0.11> 0.05). Under TEM, most of the endometrial tissue can be detected pinopodes, most fully developed in the mature stage, few in the developing stage or the withdrawal phase. Seven patients without pinopodes, kidney and blood stasis group 3 patients; 32 cases pinopodes expression of a small, nine cases of kidney and blood stasis group; 15 cases pinopodes expression medium, kidney and blood stasis group 2 cases; 6 cases cell drink expression suddenly rich, kidney and blood stasis group 1 patients; four test groups was no significant difference between using the CMH chi-square (P= 0.15> 0.05). ② different syndromes before waiting recurrent miscarriage prothrombotic state in patients with endometrial receptivity on the B-show:Measuring endometrial artery ovulation date,60 patients,15 patients between the kidney and blood stasis, four groups there was a significant difference, kidney and blood stasis group pulsatility index and resistance index were higher than the other three groups (P<0.01). In endometrial thickness, endometrial morphology, endometrial blood flow typing statistics, there was no significant difference (P> 0.05) between the four groups. ③ Correlation state of the patient on the molecular biology of endometrial receptivity before different syndromes of recurrent miscarriage thrombosis:PAI-1 was positively correlated with t-PA was (r= 0.415, P<0.01), PAI-1 and PDGF-AA was positively correlated (r= 0.390, P<0.01), PAI-1 and RI were positively correlated (r= 0.296, P<0.05), PAI-1 and endometrial blood flow typing (in accordance with the i-iii sequence coding) was negative correlation (r=-0.267, P<0.05), t-PA and VEGF was negatively correlated (r=-0.653, P<0.01), t-PA and PDGF-AA were positively correlated (r= 0.501, P <0.01), t-PA and RI were positively correlated (r= 0.399, P<0.01), PDGF-AA and RI were positively correlated (r=0.767, P<0.01), PDGF-AA and endometrial blood flow typing (in accordance with the i-order iii coding) was a negative correlation (r=-0.570, P<0.01), PDGF-AA and pinopodes (in accordance with the rich-negative sequence coding) were positively correlated (r= 0.369, P<0.01), PI and RI were positively correlation (r-0.508, P <0.01), PI and endometrial blood flow typing (in accordance with the coding sequence i-iii) was negatively correlated (r=-0.308, P<0.05), PI and HE staining (according to Yuka type-DROPOUT sequence coding) were positively correlated (r= 0.440, P<0.01), PI and pinopodes (in accordance with the rich-negative sequence coding) were positively correlated (r= 0.360, P<0.01), RI and endometrial blood flow type (according to the i-iii sequence coding) was negatively correlated (r=-0.762, P<0.01), RI and HE staining (according to Yuka type-DROPOUT sequence coding) were positively correlated (r= 0.354, P< 0.01), RI and pinopodes (in accordance with the rich-negative sequence coding) were positively correlated (r= 0.519, P<0.01), endometrial thickness and HE staining (according to Yuka type-was negatively correlated with poor type sequence coding) (r= 0.-358, P<0.01), endometrial blood flow typing (in accordance with the coding sequence i-iii) and HE staining (according to Yuka type-type differential coding sequence) was negatively correlated (r=-0.353, P<0.01), endometrial blood flow typing (in accordance with^the coding sequence i-iii) and pinopodes (in accordance with the rich-negative sequence coding) was negatively correlated (r=-0.699, P<0.01), HE staining (according to Yuka type-type differential coding sequence) and pinopodes (in accordance with the rich-negative sequence coding) were positively correlated (r= 0.332, P<0.05). VEGF results Kidney Blood Syndrome lower than the other three groups, PDGF-AA, t-PA, PAI-1 is higher than the other three groups. Wherein the PDGF-AA and t-PA were significantly different (P<0.05).3 ①before recurrent thrombotic state abortion group compared with the normal group,151 kinds of cytokines having a significant differential expression. Compared with the normal group, the disease group serum significantly increased eight kinds of cytokine expression was significantly down-regulated 143 kinds of cytokine expression by 151 cytokines abundance clustering,16 cytokine expression pattern of recurrent miscarriage were similar and different from the normal group. ② between Kidney Blood group and the normal group,118 kinds of cytokines having a significant differential expression. Compared with normal group, the Kidney Blood serum was significantly increased by 1 cytokine expression was significantly down-regulated 117 kinds of cytokine expression by more than 118 kinds of cytokines abundance Cluster analysis showed that the kidney and blood stasis group similar 76 cytokine expression patterns and different from the normal group. Between ③ Kidney Blood group and non Kidney Blood group,33 kinds of cytokines having a significant differential expression. Compared with non-Kidney Blood group in Kidney Blood serum was significantly upregulated seven kinds of cytokine expression was significantly down 26 kinds of cytokine expression by more than 33 kinds of cytokines abundance Cluster analysis showed that kidney 20 cytokine expression pattern of blood stasis group of similar, and different from the non-kidney Blood group. ④ In addition, before the recurrent thrombotic state abortion Kidney Blood group compared with the non-Kidney Blood group, in terms of cytokine signaling pathways involved in CXC subfamily, CC subfamily, Hematopoietins, PDGF family, TNFfamily, TGF-βfamily.Conclusion1 The kidney deficiency and blood stasis is the chief TCM syndrome of prethrombotic recurrent spontaneous abortions.2 Compared with other syndromes, kidney deficiency and blood stasis syndrome brings about the most prominent morphological changes in endometrial pathological tissue, which would possibly cause tense contraction of blood vessels by affecting angiogenesis system or fiber dissolution system. This will lead to a hypercoagulable state, which will stagnate blood circulation and perfusion. Glandular dysplasia on the physiological structure and deficiency in pinopodes expression will eventually lead to a decline in endometrial receptivity, and even recurrent spontaneous abortion.3 Kidney deficiency and blood stasis syndrome and non kidney deficiency and blood stasis syndrome significant differences in terms of proteomics. There are 33 kinds of cytokines with significantly different expressions, related to seven signaling pathways including CXC subfamily, the CC subfamily, Hematopoietins, PDGF, TNF, TGF-β family, etc., involving blood clotting, platelet aggregation, inflammation, angiogenesis, etc. This might be the syndrome essence of kidney deficiency and blood stasis syndrome of prethrombotic recurrent abortion.
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