| Objective:To investigate the expression of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and microvessel density (MVD) in patients with IVF during the embryo implantation window, and further clarify the value of endometrial angiogenesis and endometrial receptivity assessment by angiopoietins in predicting pregnancy outcome.Methods:A total number of twenty-five patients had been taken reproductive medical treatment at the Fourth Hospital of Hebei Medical University from August 2014 to December 2014. All the patients were at ages 20 to 35, with a normal menstrual cycle (21 to 35 days). The causes of infertility were mainly for the tubal factors, and two embryos were transplanted in fresh cycle. The endometrium tissues were collected during IVF preparation period of embryo implantation window phase, and stored in liquid nitrogen, while some of whom fixed with 4% formaldehyde. The endometrial Ang-1 and Ang-2 levels were detected using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and immunohistochemical method, while endometrial MVD was measured by immunohistochemical method.Immunohistochemical detection:The endometrium specimens were fixed with 4% formaldehyde, embedded in paraffin, and cut into 4-μm sections, mounted on glass slides, and then stained with a standard hematoxylin and eosin (H&E) immunohistochemical staining procedure for microscope analysis. PBS was usd as negative control. Five vision were randomly selected from each slice for analysis. The slides were photographed by light and fluorescence microscopy.RT-PCR technology:The endometrial tissues were taken from all patients. Total RNA was extracted from different samples according to the manufacturer’s protocol for Trizol Reagent. Then, complementary DNA (cDNA) was synthesized from 2μg of total RNA using a first-strand cDNA synthesis kit, GAPDH of 605bp length was as internal reference. PCR cycling conditions of Ang-1 and Ang-2:95℃ 10min, 1cycle; 95℃ 45s,60℃ 40s, 72℃ 50s,35cycles; 72℃ 10min,1 cycle. Products were run 1.5% agaros gel electrophoresis, then photographed and image analyzed by VDS system. IOD values were adjusted by internal reference, then the adjusted values were for statistical analysis.Controlled ovarian hyperstimulation:give acetate triptorelin (Beijing Boente Pharmaceutical Co Ltd) 1.3mg during 6-7 days after ovulation (mid-luteal), then give Recombinant Human Follitropin Alfa Solution for Injection (Merk Serono Pharmaceutical Factory, Italy) 150-225IU when it reached the down-regulation standard. We monitored the follicular growth and endometrial changes by transvaginal ultrasound. When the diameter of follicle (≥2 follicles) was bigger than 20mm, the patient received injection of Azeroth (Recombinant Human Choriogonadotropin Alfa Solution for Injection, Italy) 250μg. Thirty-seven hours later, to obtain oocytes and fertilized in vitro. Three days after ovulation, we transplanted two embryos. Twenty-eight days after transplantation, see the gestational sac by type-B ultrasonic examination was considered as clinical pregnancy.All data were analyzed using SPSS 13.0 statistical software. Data were presented as x±s. The comparisons among various groups were performed using t test. A P value of 0.05 was considered as statistically significant.Results:1 Comparison of general situations between the pregnant group and the non-pregnant groupComparing the differences between the pregnant group(n=12) and the non-pregnant group(n=13):there were no statistical significance (P>0.05) on the patients age, infertility years, infertility factors, base FSH, base LH and AFC.(Tablel)2 Comparison of IVF-ET conventional treatment between the pregnant group and the non-pregnant groupComparing the differences between the two groups:there were no statistical significance (P>0.05) on the oocyte retrieval rate, good quality embryo rate, endometrium thickness, E2 and P on the ET day. (Table2)3 The expression of Ang-1, Ang2 and MVD examined with immunohist ochemical staining3.1 Angl was expressed at endometrial stromal cells, glandular epithelial cells and vascular endothelial cells in the preliminary period during the embryo implantation window phase. The pregnant group (n=12), the expression "-" 1 cases, "+"2 cases,"++"5 cases,"+++" 4 cases. Non pregnancy group (n=13), the expression "-" 4 cases, "+" 5 cases, "++"3 cases, "+++"1 cases, (P =0.026). (Table3)3.2 Ang2 was mainly expressed in endometrial stromal cells and glandular epithelial cells. Ang-2 was expressed in the pregnant group and non-pregnant group, pregnant group (n=13), the expression "-" 2 cases, "+"3 cases, "++"5 cases, "+++" 2 cases. Non-pregnancy group (n=13), the expression "-" 3 cases, "+"6 cases,"++"2 cases,"+++"2 cases, (P=0.3335). (Table4)3.3 CD34 was specificity expressed in the endothelial cells, microvessels were riched expressed in endometrial tissue during embryo implantation window phase. The quantity of pregnancy group’s expression of MVD (16.42+4.66) was obviously more than non pregnancy group’s (12.31+4.40), the difference was statistically significant (P<0.05). (Table5)4 The expression of Ang-1 and Ang2 examined by RT-PCR4.1 The expression of Ang-1 mRNA of endometrium in pregnancy group was significantly higher than that in non-pregnancy group in embryo implantation window period, the expression of Ang-1mRNA in pregnancy group is 0.72+ 0.35 (n=12), the expression in non-pregnancy group is 0.33+0.32 (n=13), the difference was statistically significant (P=0.008). Higher expression of Angl mRNA made endometrial receptivity become more better and more conducive to embryo implantation. (Table6)4.2 Expression of Ang-2mRNA in pregnancy group was 0.65+0.60 (n=12), in non pregnancy group was 0.65+0.41 (n=13), there was no significant difference (P=0.988). (Table6)5 The corelation analysis of Ang-1、Ang-2 and MVD on the expression level of protein5.1 The expression of Ang-1 and MVD in patient with IVF-ET in enbryo implantation window phase, which correlation coefficient was 0.533, P =0.006, the difference was statistically significant. (Table7)5.2 The expression of Ang-2 and MVD in patient with IVF-ET in enbryo implantation window phase, which correlation coefficient was 0.268, P =0.166, no significant difference. (Table8)Conclusions:1 Ang-1 was expressed in stromal cells, glandular epithelial cells and vascular endothelial cells in endometrial in the preliminary period of embryo implantation window phase.2 Ang-1 mRNA might be one of the genes related to endometrial receptivity. Low expression of Ang-1 mRNA in endometrial of IVF patients during embryo implantation window phase might be one of the reasons causing the low endometrial receptivity.3 Ang-2 was mainly expressed in endometrial stromal cells and glandular epithelial cells in the preliminary period of embryo implantation window phase.4 The expression of Ang-2 mRNA in endometrial of IVF patients in the preliminary period of embryo implantation window phase had no statistical difference between pregnancy group and non pregnancy group, which roles of the assessment of endometrial receptivity needs further study.5 MVD in endometrium of IVF patients in pregnancy group was higher than that of non pregnancy group in the preliminary period of embryo implantation window phase, which difference was significant. Small blood vessels with fuction were richer in the endometrium of patients in pregnancy group, which might be related to its strong angiogenesis ability. The abundant blood flow is more advantageous to embryo implantation.6 Ang-1 was a promoting factor of angiogenesis and rich endometrial blood flow perfusion is beneficial for embryo implantation. Correlation analysis indicated that expression of endometrial Ang-1 protein was significantly correlated with MVD in embryo implantation window phase, while Ang-2 protein expression had no correlation with MVD. |
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