| Objective: With the developing of assisted reproductive technology, the pregnancy rate of IVF has increased from 20%-30% to about 50% quickly. The success of embryo implantation mainly depends on embryo quality and endometrial receptivity. On the basis of top quality embryos, the establishment of endometrial receptivity is the key point to the process of implantation. The endometrial receptivity maybe closely associated with the factors which regulute the endometrial microvascular and blood supply. Among the numerous factors associated with angiogenesis, we selected endothelial nitric oxide synthases(eNOS) to study.eNOS is an enzyme that convert the amino acid L-arginine to citru lline and NO.eNOS plays an important role in regulating the blood supp ly of endometrium. The product—NO is one of an important multiple f untional factors that functions as a major mediator of numerous biologic al processes and plays a role on biology, including regulate vascular blo od pressure,participate in cell damage repair, signal transduction and so on. The expression of eNOS in the endometrium may closely associate d with the vascular blood supply,in that to influence the endometrial re ceptivity.Angiogenesis could be evaluted by MVD. The personality of MVD is that endothelial cells could be dyed by CD34 antibody tan,so we could use that to observe the angiogenesis of microvessel[2]. MVD was used to evaluate angiogenesis in organization, the higher the eNOS expressed, the richer the new capillaries grew. The blood flow can be reflected by ultrasound monitoring parameters including pulsatility, resista nce index and the ratio of systolic and diastolic blood flow.So the expression of eNOS, the expression of eNOS and the blood flow parameters constituted to a series indicators that assessed the endometrial receptivity of patients who would undergo the assisted reproduction treatment.Methods: A total of 60 patients who would undergo the assisted reproduction treatment first from The fourth hospital of Hebei University from Augst 2014 to June 2015 were selected. Inclusion Criteria: patients with normal menstrual cycle, aged between 20 to 35 years and with simple tubal factor. Endometrium was collected in the implantation window phase before IVF.We used immunohistochemistry and reverse transcription polymerase chain reaction to detect the expression of eNOS and MVD.1 collect endometrial samples: Endometrium was collected in the natural menstrual cycle before controlled ovarian stimulation. Firstly, monitoring follicles while we used semi-quantitative determination of test urine luteinizing hormone at the same time. When the urinary LH reached above 20 IU/L,gave HCG to the patient and recorded endometrial thickness and morphology. Ovulation was judged When the follicle disappeared or significantly narrowed. Endometrial blood flow, morphology and blood flow parameters RI?PI?S/D were detected by GE Voluson E8 color doppler ultrasonic diagnostic apparatus at 6~7 days after ovulation. The test completed by only specialist one. The average of the 3 times continuous measurement value was caculated.Endometrial samples were collected by disposable endometrial sampler. One part was storaged at- 80 ?refrigerator, another part in formaldehyde fixed fluid. Nobody had abnormal vaginal bleeding.2 Controlled ovarian stimulation: leuprorelin Acetate Microspheres(Gonadotrophin-releasing-hormone agonist,Gn RH-a,China burns) 1.4 mg was injected at 6~7 days after ovulation. gonadotropin(r- FSH, Merck Serono)150-225 IU were given when it reached at down-regulation standard. Then using ultrasonic to monitor the endometrium thickness and follicular growth situation. When the diameter of follicles(?2 follicles) were bigger than 20 mm, the patient received injection with vidrel(Merck Serono) 250 ug. About 36~37h later,to get ovum pick-up, added sperm to the culture dish in time,then fertilized in vitro. We transferred 2 embroys after 3 days. 28 days after transplantation, we used type-B ultrasonic to examine gestational sac,which was considered as a signal of clinical pregnancy. According to the clinical pregnancy or not, patients were divided into pregnancy group and non-pregnancy group. There were 30 people in pregnancy group and 30 people in the other group.3 RT-PCR: The expression of eNOS mRNA in endometrium were detected. Total RNA was extracted first. Join Tri Quick total RNA extraction Reagent in the study group and control group respectively, grinded endometrial tissue on ice, added the same endometrial tissue and Rezol 1ml to the homogenate.When total RNA was extracted, then reverse transcription to obtain cDNA, and then PCR amplification was crarried out. Amplified fragment length was 340 bp, and at the same time an internal reference with the length of 605 bp GAPDH was amplified. PCR cycle conditions: PCR amplification conditions: 94 with ?10 min for one cycle(94?45 s, 54 30 ?s, 72?50 s) 35 cycles, 72 for 7 min, 4 heat preservation. Products wer? ? e run by 2% agaros gel electrophoresis, then photographed and image analyzed by gel-proanalysis3.1system. IOD values were adjusted by internal reference, then the adjusted values were for statistical analysis.4 Immunohistochemical detection: Endometrial specimens were cut i nto 4?m sections after then fixed and embedded by paraffin. Then HE and SP immunohistochemical staining methods were performed respectiv ely. Dewaxing, high-pressure antigen repair, closed, polyclonal rabbit anti people eNOS(diluted concentration of 1:100), two resistance, DAB col or, counterstained with hematoxylin, dehydration and then transparent, ne utral gum sealing piece. Use PBS instead of a primary antibody as neg ative control. Observed by a microscope and photographed.MVD criterion used rabbit anti CD34 staining, dilute the concentrat ion of 1:100, used immunohistochemical SP method and stepped with th e former.We calculated MVD according to the judgment standard of Wei dner.5 Statistical methods Statistical analysis was performed with SPSS version 13.0 software. Quantitative data were expressed as mean±standa rd deviation((?)+ s), statistical analysis was performed using analysis of student t-test. The statistical analysis of the ranked data was performed by rank-sum test.Probabilily values less than 5% were considered to be significant. The correlation between two variants was analysised by Spe arman rank correlation. Probabilily values less than 5% were considered to be significant.Results:1 Comparing the differences between the pregnant group(n=30) and the non-pregnant group(n=30). There were no statistical significance(p>0.05)on the patients age, infertility years, b FSH(basic follicle stimulating hormone), BMI, the day of Gonadotrophin costs,the dosage Gonadotrophin costs and the number of oocytes.2 The expression of eNOS mRNA of endometrium in pregnancy group was significantly higher than that in non-pregnancy group during embryo implantation window period, the expression of eNOS mRNA in pregnancy group was( 0.72±0.21), the expression in non-pregnancy group was(0.29±0.12).The difference was statistically significant(p<0.05).3 The eNOS protein expressed at glandular epithelial cells and endometrial stromal cells in both pregnancy group and non-pregnancy group. Pregnancy group(n=30), the expression "-" 1 cases, "+" 5 cases, "++" 12 cases, "+++" 12 cases. Non-pregnancy group(n=30), the expression "-" 12 cases, "+" 14 cases, "++" 2 cases, "+++" 2 cases, The difference was statistically significant(p<0.05).4 The MVD counts in pregnancy group was( 14.53±3.28), the expression in non-pregnancy group was(12.20±3.83).The difference was statistically significant(p<0.05).5 Correlation analysis of the eNOS expression and MVD Analysised by Spearman rank correlation,there was a positive correlatio n between the eNOS expression in endometrium and MVD counts(r=0.814,P<0.05).6 The pulsatility index?resistance index and systolic/diastolic of the endometrium in pregnancy group were lower than that in the non- pregnancy group. The difference was statistically significant. The endometrial blood flow in pregnancy group was more abundant than that in non-pregnant group.Conclusion :1 The expression of eNOS mRNA and protein in pregnancy group was up-regulated than that in non-pregnancy group,that eNOS could help to improve the endometrial receptivity and increase embryo implantation chance. The eNOS was approved to be one of the factors that related to endometrial receptivity.2 The increasing with MVD confirmed that could improve the endometrial receptivity.3 There was a positive correlation between the eNOS expression and MVD. So we inferred that eNOS played an important role in the angiogenesis of endomrtrium.4 The pulsatility index?resistance index and systolic/diastolic of the endometrium in pregnancy group were lower than that in the non- pregnancy group.The blood supply was richer than the non- pregnancy group.The degrees of richness of blood flow was closely related to the outcome of pregnancy. |
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