| BackgroundUterine receptivity is one of crucial factors in human reproduction. The human endometrium undergoes series of morphologic and functional changes in order to provide the most suitable conditions for embryo implantation. The endometrium is only receptive for an embryo in a limited time span, which is called Implantation Window and regarded to be on days 5-9 postovulation (Bonilla-Musoles et al.2013). There are various methods to assess the endometrial receptivity, such as transvaginal ultrasonography, endometrial biopsy and immunohistochemical analysis.However, endometrial biopsy is an invasive method and immunohistochemical analysis is complicated and expensive, they are not accepted widely in clinical practice. It becomes a merge to find a method that is non-invasive and convenience to assess endometrial receptivity. Therefore, transvaginal ultrasonography may serve as such an ideal non-invasive tool.Since angiogenesis, (sub-)endometrial vascularization and perfusion were found to play critical roles during embryo implantation, many researchers began to focus on the quantitative assessment of (sub-)endometrial blood flow. The conventional color Doppler, pulsed Doppler and the novel 3D-PDA (three-dimensional power Doppler angiograpghy) have been used to assess (sub-) endometrial blood flow. However, conflicting results have been reported. For basal arteries and spiral arteries are tiny vessels with low velocity of blood flow and different ultrasonic instruments have different sensitivity for these tiny vessels, the method to assess endometrial and subendometrial perfusion has not yet been established definitely.With the advent of second-generation contrast agents, which can visualize the capillary net of the examined tissue, CEUS (contrast-enhanced ultrasound) has been widely used in evaluating blood flow perfusion of various organs. Since the diameter of the contrast agent bubbles were much smaller than red blood cells, CEUS with the contrast agent could greatly improve the detective level of tissue microvascularization, even when the microvessel diameter was smaller than 40um. Thus, CEUS might be a better way to investigate the microcirculation of the endometrium. This was proved during early implantation in the macaque uterus, but not in human beings. Among the main CEUS parameters, Pi and AUC were indications of enhancement intensity and were applied to evaluate blood perfusion.At present, the whole world there are about 13%-17%of childbearing age couples suffering from infertility or sterility, infertility accounted for about 15%-30%. Previous studies found that impaired uterine perfusion might be a cause of unexpained infertility and (sub-)endometrial perfusion played critical roles during embryo implantation. Thus a population of unexplained infertility was selected to represent a suboptimal endometrial receptivity in this study. The aim of this study was to evaluate uterine receptivity between the normal fertility and unexplained infertility during natural cycles by the assessment of endometrial and subendometrial perfusion using CEUS and tried to find out which index would be better:Pi (peak intensity) or AUC (area under the curve).Chapter OneAssess endometrial and subendometrial perfusion by using CDFIObjectiveTo evaluate the clinical utility of CDFI to assess endometrial and subendometrial perfusion, taking MVD as the gold standard.Materials and methods1 Participants30 fertile women and 30 unexplained infertile women were recruited into this study.15 women with normal fertility and 18 women with unexplained infertility accepted endometrial biopsy, and the rest declined this procedure. The study was approved by the Ethics Committee, and patient consent was obtained.The inclusion criteria of the study group were as follows:patients, who were unable to conceive after 1 year of regular unprotected intercourse, were admitted. The diagnosis of unexplained infertility was made according to the guidelines published by The Practice Committee of the American Society for Reproductive Medicine (ASRM).The inclusion criteria of the control group were as follows:women with a history of a normal delivery in a year and with no specific gynecological complaints or check-ups and no breastfeeding in three months.Besides, all the participants from two groups should have regular menstrual cycles of 26-32 days and had not taken any medication that could possibly interfere with the pelvic blood supply or used hormones in three months prior to the study. Coagulation deficiency or hemorrhagic disorders were ruled out among them. All the participants are required to use non-drug contraception in the period of testing.2 Ultrasound examination and endometrial biopsy date settingUltrasound examination (CDFI and CEUS examination orderly) and biopsy were both performed on D10-D12 (late-proliferative phase), LH-LH+2 (ovulation period) and LH+8-LH+10 (Implantation Window) of a menstrual cycle and ultrasound examinations were performed prior to biopsy. All of the ultrasound scans and measurements were performed between 9am and 12am. The date of ovulation was confirmed by transvaginal ultrasonography and urinary luteinizing hormone (LH). After the first CEUS examination and biopsy on D10-D12 of menstruation, patients were told to have their urinary LH tested twice daily (10 a.m. and 10 p.m.) by using LH diagnostic kit (Hemtrue, Shanghai Kaichuang Biological Technology CO.LTD.) until LH surge day (with the occurrence of T>C). Transvaginal ultrasonography was then performed daily to monitor the follicular rupture since the LH surge day. Only when the follicular rupture actually occurred on LH-LH+2, did the study continue during the time of the implantation window.3 CDFI examinationA Phillip IU22 ultrasound system (Phillips, Holland), a C8-4v transvaginal transducer (5-7.5MHz) was used. A true longitudinal view of the uterus was first obtained on the static grey-scale images, and color Doppler mode was then activated to assess the endometrial-subendometrial blood flow on a longitudinal scan of the uterus. The color sample frame was placed including all of the endometrium. The velocity scale was set as 2.1cm/s and color gain was set as 80%±2%. According to the distribution pattern of endometrial-subendometrial blood flow, the endometrial-subendometrial blood flow was classified into 3 types:Type A, visualized vessels penetrating into the endometrial region and approaching the uterine cavity;Type B, visualized vessels penetrating into the endometriral region but not beyond 1/2 of the monolayer endometrial thickness; Type C, visualized vessels penetrating into the subendometrial region but not entering the endometrial region.4 Endometrial biopsyThe participant was told to drink plenty of water to keep the bladder full and lie in the bladder-lithotomy position. After the routine disinfection, a disposable uterine tissue suction tube (LILYCLEANER, Shanghai Jiabao Medical Health Care Technology CO.LTD.) was used. The average depth of intake was 7.8 cm. When reaching the posterior (or anterior) wall of the fundus, the tube was drawn back and a small amount of endometrial tissue was brought out by negative pressure (≥150mmHg).5 ImmunohistochemistryThe endometrial tissues removed were embedded in paraffin, sectioned, and immunohistochemically stained for CD34 with a monoclonal mouse anti-human antibody (QBEnd-10,Gene,China). A positive control (known ovarian cancer section) and a negative control (PBS instead of primary antibody) were used for all stains. Positive CD34 staining was seen in the cytoplasm of vascular endothelial cells. Brown-stained individual vascular endothelial cells or endothelial cell clusters were counted as one microvessel. Vasculature with significant smooth muscle wall or that with more than eight red blood cells was not counted. The MVD (microvessel density) was determined by the modified quantification method as reported by Weidner et al. Five fields of the most MVD expression particles were observed at low magnification (x 100), and then counted at higher magnification (x 200). The mean MVD value was calculated.6 Statistical analysisSPSS 13.0 statistical software was used for data analysis. Quantitative data was expressed as (x±s). An independent sample t-test was used to compare the quantitative data and Mann-Whitney U test was to compare the ranked data between the two groups. Kruskal-Wallis test was used to compare the ranked data among the multi-phases of menstrual cycle. p< 0.05 was considered statistically significant.Results1 General characteristics of the study population30 patients (23 to 32 years old; average age:28.81±2.59 years old) were recruited into the study group. All of them finished the CEUS examination and 18 of them (23 to 32 years old; average age:27.28±2.87years old) finished the endometrial biopsy.30 women (20-31 years old; average age:27.14±2.01 years old) were recruited into the healthy control group. All of them finished the CEUS examination and 15 of them (21 to 31 years old; average age:25.40±3.46 years old) finished the endometrial biopsy. There were no significant differences of age between two groups (p=0.075,p=0.098).2 Distribution pattern of endometrial-subendometrial blood flow with CDFI2.1 Comparison of distribution pattern of endometrial-subendometrial blood flow between two groupsIn the late-proliferative phase, the control group had a better endometrial-subendometrial perfusion than the study group (ARcontrol group= 34.83, ARstudy group=26.17;p=0.040). There were no significant differences of distribution pattern of endometrial-subendometrial blood flow between two groups either in the ovulation period or in Implantation Window (p=0.092,p=0.769)2.2 Comparison of distribution pattern of endometrial-subendometrial blood flow among multi-phases of menstrual cycle in two groupsThere was no significant difference of distribution pattern of endometrial-subendometrial blood flow among multi-phases of menstrual cycle either in the study group (p=0.092) or in the control group (p=0.769).3 Endometrium MVD3.1 Comparison of MVD between two groupsThe control group was found to have a higher MVD than the study group in the late-proliferative phase (15.31±2.28 vs.11.77±2.62; p<0.001) and the ovulation period (11.94±1.71 vs.7.39±1.12;p<0.001). No significant differences were found between the two groups in Implantation Window (13.64±1.75 vs.13.23±2.57; p=0.607).3.2 Comparison of MVD among multi-phases of menstrual cycle in two groupsA significant differences was found among multi-phases of menstrual cycle both in the study group (p<0.001) and in the control group (p<0.001). Thereinto, MVD showed significances between each of the two phases (p<0.001).Conclustion1 MVD is the most direct reflection of angiogenesis. The more microvessels are, the richer perfusion is. MVD is the quantified index of angiogenesis. According to the result of MVD, it was found that there were richer microvessels in the healthy fertile women than in the unexplained infertile women, both in the late proliferative phase and the ovulation period. It is suggested that a damaged endometrial and subendometrial perfusion might be one of the causes of unexplained infertility.2 According to the result of CDFI in this study, the control group had a better endometrial-subendometrial perfusion than study group in the late-proliferative phase, whereas MVD indicated that the control group had a richer uterine microvascularization than the study group in both late-proliferative phase and the ovulation period. No significant difference of endometrial-subendometrial blood flow distribution pattern was found among the multi-phases of menstrual cycle in either the group or the control group while there was a significant difference of MVD among the multi-phases of menstrual cycle in both groups.,mainly because of the high dependence of gain and other machine settings, attenuation and reproducibility. The reason for the discordance between the results of CDFI and MVD may be as followed:1) the limitation of CDFI to assess vascularization; 2) Though it is thought that there is slight harm to the endometrium with the biopsy, the biopsy might acivate the angiogenesis, which resulted in reducing the difference of endometrial and subendometrial perfusion between two groups. CDFI could not detect the difference however. Thus we come to a conclusion that CDFI could not precisely assess the endometrial and subendometrial perfusion.Chapter TwoAssess endometrial and subendometrial perfusion by using CEUSObjectiveTo evaluate the clinical utility of CEUS to assess endometrial and subendometrial perfusion, taking MVD as the gold standard.Materials and methods1 ParticipantsThe same as Chapter One.2 Ultrasound examination and endometrial biopsy date settingThe same as Chapter One.3 Preparation of suspension liquid of contrastThe seconed generation contrast, SonoVue (Bracco, Milan, Italy) was used. Check and ensure container and cap is sound before use.5 ml of 0.9% saline solution was injected into the bottle and shaken for 20 seconds until the completely dispersed and homogeneous milky liquid occurred.4 CEUS examinationA Phillip IU22 ultrasound system, a C8-4v transvaginal transducer (5-7.5MHz) and a microbubble US contrast agent, SonoVue (Bracco, Milan, Italy) were used. An automatic contrast-enhanced mode with MI (mechanical index)< 0.06 pulse inversion harmonicsand was selected. The whole procedure of CEUS examination was carried out under a double-dual mode. The probe was placed in the plane of the central longitudinal view of the uterus to visualize the endometrial and subendometrial area. The agent was shaken for about 20 seconds with 5 ml of 0.9% saline solution and 2.4 ml of this suspension was injected into a cubical vein as a bolus, followed by a 10ml normal saline fluid. Then the CEUS examination was started and the serial images ranging from the beginning to 2 min were obtained and recorded for the retrospective evaluation of time-intensity curve (TIC). All ultrasonographic examinations were carried out by the same experienced ultrasonographer.5 TIC analysisTIC was performed using the Q-Lab software for ultrasound analysis. The procedures were as follow:(1) Choosing the regions of interest (ROIs). All ROIs were drawn by one investigator to mitigate variation and analyzed during the same time course. In this investigation, endometrial region (2 regions) and subendometrial region (6 regions) were chosen and labeled as sub-region 1-8 respectively. Thereinto, subendometrial region was defined as 5 mm surrounding the endometrial borders. (2) Recording and normalizing the sustained curves by using the LDRW WIWO normalization (120 s). The parameters AUC and Pi of all the sub-regions were given automatically. (3)The average Pi and AUC value of 1-2 sub-regions and 3-8 sub-regions were calculated.6 Endometrial biopsyThe same as Chapter One.7 ImmunohistochemistryThe same as Chapter One.8 Statistical analysisSPSS 13.0 statistical software was used for data analysis.Quantitative data was expressed as (x±s). An independent sample t-test was used to compare the quantitative data and Mann-Whitney U test was to compare the ranked data between the two groups. Analysis of variance (ANOVA) was used to compare the quantitative data and Kruskal-Wallis test was used to compare the ranked data among the multi-phases of menstrual cycle. The CEUS parameters and MVD were compared using bi-variant correlation analysis. LSD test was used for the pairwise comparison of quantitative data. p< 0.05 was considered statistically significant.Results1 General characteristics of the study populationThe same as Chapter One.2 Evaluation of uterine microcirculatory perfusion with CEUS2.1 Comparison TIC parameters between two groupsIn the comparison of the TIC parameters between the two groups in multi-phases, it could be seen that in the late-proliferative phase, control group had a significantly higher endometrial Pi (p<0.001) as well as subendometrial Pi (p<0.001) and AUC (p=0.004) than the study group. In the ovulation period, the control group had a significantly higher endometrial Pi (p<0.001) and AUC (p=0.021) as well as subendometrial Pi (p<0.001) and AUC (p=0.003). In Implantation Window, no significant differences were found between two groups.2.2 Comparison of TIC parameters among multi-phases of menstrual cycle in two groupsIn the study group, neither the endometrial Pi and AUC nor the subendometrial AUC differed among the multi-phases of menstrual cycle (p=0.134,p=0.096, p=0.059). However, subendometrial Pi showed significant differences among multi-phases (p<0.001). It decreased from the late-proliferative phase, reached a nadir in the ovulation period and peaked in the implantation window. Thereinto, subendometrial Pi significantly differed between the late-proliferative phase and the ovulation period and so did it between the ovulation period and the Implantation Window. But there was no significant difference of subendometrial Pi between the late-proliferative phase and Implantation Window.In the control group, neither endometrial Pi and AUC nor subendometrial AUC differ among the multi-phases of menstrual cycle (p=0.340, p=0.120, p=0.779). However, subendometrial Pi showed significances among multi-phases (p<0.001). It peaked from the late-proliferative phase, reached a nadir in the ovulation period and increased until Implantation Window. Thereinto, Pi showed significances between each of the two phases.3 Endometrium MVD3.1 Comparison of MVD between two groupsThe same as Chapter One.3.2 Comparison of MVD among multi-phases of menstrual cycle in two groupsThe same as Chapter One.4 Correlation between subendometrial Pi and MVDMVD were positively correlated with subendometrial Pi in the study group and control group respectively (r=0.806,r=0.733).Conclustion1 In reviewing the CEUS TIC parameters, the control group had a higher endometrial Pi as well as subendometrial Pi and AUC than the study group in the late-proliferative phase. In the ovulation period, the control group had a higher endometrial Pi and AUC as well as subendometrial Pi and AUC than the study group. Meanwhile, MVD was significantly higher in the control group than in the study group in the late-proliferative phase and the ovulation period. It indicated that CEUS had an advantage on assessing the endometrial and subendometrial perfusion compared with CDFI.2 It was found that there was no significant difference in the change of endometrial Pi, AUC or subendometrial AUC throughout the menstrual cycle. Only the subendometrial Pi showed significant periodic changes in menstrual cycle and had a positive correlation with MVD in both groups. The positive correlation between subendometrial Pi and MVD was in agreement with the findings of the previous researches on tumor. It suggested that subendometrial Pi might be the most sensitive index to access the endometrial microcirculatory perfusion compared with endometrial Pi, endometrial AUC and subendometrial AUC.3 Subendometrial Pi and endometrial MVD in the study group were significantly lower than those in the control group during a menstrual cycle, except in the Implantation Window. The hypothesis is that the difference between unexplained infertility and healthy fertility may have occurred before Implantation Window. A defective endometrial blood supply before the Implantation Window may be a cause of unexplained infertility.4 This study offered us information on the characteristics of the normal and abnormal microvascular changes throughout the menstrual cycle. In the control group, subendometrial Pi peaked during the late-preliferative phase and reached a nadir in the ovulation period, and then increased until Implantation Window. However, in the study group, the subendomerial Pi peak was seen in Implantation Window. Therefore, we supposed a failure in well-prepared uterine blood flow in the late-proliferative phase may lead to a poor development of endometrium and the subsequent infertility. |
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