Endometrial Microvessel Density And Three-dimensional Ultrasonography And Power Doppler Angiography Parameters Assess Endometrial Receptivity

BACKGROUNDSuccessful implantation depends on a close interaction between the functional blastocyst and the receptive endometrium.Under certain conditions,we can not improve the quality for embryos significantly,we turn to improve the endometrial receptivity.Histological evaluation, now considered to add little clinically significant information,should be replaced by functional assessment of endometrial receptivity.Angiogenesis,the formation of new blood vessels from existing vessels, plays a critical role in various female reproductive processes such as develoment of a dominant follicle,formation of a corpus luteum;growth of endometrium and implantion. A good blood supply towards the endometrium is usually considered as an essential requirement for implantion. Angiogenesis can be described by a microvessel density(MVD) count. MVD can be measured by staining for CD34. CD34 is a glycosylated transmembrane protein present on endothelial cells and may possibly be involved in endothelial migration during angiogenesis.Potential uterine predictors for implatation that are measurable by sonography include endometrial thickness and volume,endometrial pattern and also blood flow in the uterine and (sub)endometrial arteries.Unfortunately,two-dimensional ultrasound studies on blood flow in (sub)endometrial vessels are limited in their selection of individual vessels and the assumption that these are representative of the subendometrium as a whole. The combination of power Doppler sonography with the three-dimensional ultrasound (3D-PDS) is better suited to the study of the (sub)endometrial vasculature, as it provides a unique tool for examining the blood supply of the whole (sub)endometrial region, as opposed to individual vessels or two-dimensional planes.Ultrasound parameters including:endometrial volume,endometrial thickness, endometrial pattern, uterine artery Doppler pulsatility index (PI) and resistance index (RI),three-dimensional ultrasound parameters such as vascularization index (VI), which measures the ratio of the number of colour voxels to the number of all the voxels, is thought to represent the presence of blood vessels (vascularity) in the endometrium, and this was expressed as a percentage (%) of the endometrial volume;flow index (FI), the mean power Doppler signal intensity inside the endometrium, is thought to express the average intensity of flow;vascularization flow index (VFI) is a combination of vascularity and flow intensity.Previous studies could not demonstrate that the endometrial volume is predictive for pregnancy, and this could be explained by methodologic differences in the volume calculation. A triple-layer endometrial pattern and an endometrial thickness greater than 7 mm have been proposed as markers of endometrial receptivity but have yielded a high percentage of false-positive results.Three-dimensional ultrasonography and 3DU-PDA may predictive IVF-ET outcome by the way of evaluating endometrial receptivity- uterine cavity micro-environment. Chen Lei-ning found the relationship between three-dimensional ultrasonography and power Doppler angiography (3DU-PDA) parameters of endometrium on HCG administration day and on-going pregnancies or early spontaneous abortions after good quality embryos transfer,they found that the the endometrial vascularization index, flow index andvascularization flow index were significantly higher for on-going pregnancy group than the early spontaneous abortion group(P< 0.05).Pinopods are bleb-like protrusions on the apical surface of the endometrial epithelium.These structures are several micrometers wide and project into the uterine lumen above the microvilli level. They were first described in mice, and later in human endometrium.The term'pinopod', from the Greek'drinking foot', signifies their pinocytotic function in the mouse.Nikas fond that pinopods can be only fond between days 20 and 21 of a regular menstrual cycle,and that their total lifespan did not exceed 48 h. More and more scholars think that uterine pinopodes as markers of the'window of implantation' According to expression pinopodes endometrial lining of the percentage of the total, is divided into rich (50%), moderate (20%～50%) and a small amount.Pinopodes were classified as developing,fully developed,orregressing.More and more studies evidenced that pinopodes is the exact marker of implantation window.OBJECTIVEThe aim of this study was to evaluate the relationship between microvessel density and endometrial and subendometrial blood flows measured by 3D power Doppler ultrasoud and their role of assessing endometrial receptivity.METHODS1,Patients Patients undergoing their frist IVF/ICSI-ET cycle in the Assisted Reproduction Unit of the Department of Obsterics and Gynaecology,The University of Southern Medical University between April 2009 to December 2009 were recruited if they meeting the inclusion and exclusion criterias. Finally, a total of 104 patients were recruited, the average age is 31.64±4.09 years old.2,The inclusion criteria①All the patients have enough intelligence to participate the study,and every patient gave a written informed consent prior to participating in the study.②A mid-luteal phase long protocol③Women with regular menstrual cycles④Fresh cycleThe exclusion criteria①Acute cervicitis;vaginitis;bacterial vaginosis;Bartholin's duct cyst and abscess;urinary tract infection and other pelvic inflammatory disease②Blood diseases and disorders③Failue to embryo transfer④Luteinized unruptured follicle syndrome(LUFS)⑤Application of estrogen,aspirin, salvia, at least a month before the the assisted cycle3. SubgroupThe patients in every part of this study were divided into two groups:the clinical pregnancy group; non-pregnant group.4. IVF protocolAll subjects had ovarian stimulation according to a mid-luteal phase long p rotocol.Follicular monitoring by vaginal ultrasonography was initiated on cycle day 10, and after a dominant follicle sized 14mm was identified, we detection the urinary LH surges every 6 hours daily by urinary luteinizing hormone plate (Chemtrue company,ShangHai). Blood for LH and E2 determination was drawn on the day of the urinary LH surge.36 hours later, vaginal ultrasonography was initiated,if the leading follicle (s) disappeared we gave the patients GnRHa 7 days later.5.Gain endometrium tissueOn the GnRHa day we gain endometrium tissue by disposable uterine cavity suction catheter (C3.1/30-1S, Jiabaocompany,ShangHai) The catheter entered uterine cavity about 7.8cm, aspirating two times. All of the endometrium tissue were divided into two groups,one in formaldehyde(10%) for Imunohistochemistry(IHC); one in glutaraldehyde(2.5%)for scanning electron microscope(SEM).6.ImmunohistochemistryEndometrial microvessel density(MVD) was detected by dyeing vascular endothelial cells with CD34 mouse anti-human monoclonal antibody.7.Three-dimensional ultrasound observation time and Ultrasonic InstrumentAll ultrasound measurements were performed by one ultrasound doctor on the day of GnRHa and HCG. throuth Voluson i (GE Medical Systems).8. Scanning electron microscope(SEM)Hitachi,S-3000N,Japan (AV:0.3-30kV; 2e-image resolution:3nm; Magnification:×15～300000)9. Image analysisMVD count by Image J software (U.S. National Institutes of Health), and the software can calculate immunohistochemistry images brown, size of the area percentage of the total field of vision automately.10. Statistical analysisStatistical analysis was performed using the Statistical Program for Social Sciences (SPSS Inc., version 13.0, Chicago, IL). Statistical.Comparison was carried out by Independent-Samples T test and Chi-Square test. The receiver operating characteristic (ROC) curve analysis and Logistic regression were applied to determine the best predictive variables.The two-tailed value of P<0.05 was considered statistically significant.RESULTS1. Scanning electron microscopy observation endometrial pinopods The clinical pregnancy group (3 cases); non-pregnant group (4cases).we can see pinopods both of the two groups,so we can say that we get the endometria in the period of window of implantation.2.MVDThe MVD for the clinical pregnancy group (50 cases,4.12±1.84%)were significantly higher than non-pregnant group (54 cases,3.46±1.26%)(t=-2.127, P=0.036).3. Three-dimensional ultrasound parametersThe clinical pregnancy group (31 cases), non-pregnant group (36 cases).There was no significant difference in 3DU-PDA parameters on GnRHa day between the two groups, VI:2.24±4.11%/1.47±1.63%; FI:25.26±5.85/23.41±4.41; VFI: 0.72±1.18/0.44±0.509; subendometrial VI:4.18±4.32%/3.20±3.24%; subendometrial FI:25.99±6.20/25.15±4.08; subendometrial VFI:1.19±1.36/1.00±0.99; endometrial volume:3.80±1.84ml/3.75±1.79ml, The patients of the clinical pregnancy group on HCG day in subendometrial FI (27.56±4.89) is higher than that of the non-pregnant group (25.15±4.08%), the difference was statistically significant (t=-2.398, P= 0.019).CONCLUSIONIn the IVF/ICSI-ET cycles, microvessel density and 3DU-PDA parameters is helpful to assessing endometrial receptivity,but the specificity is low.