| ObjectivesDiscuss the accuracy and practicability of positive contrast agents combined with negative contrast agents in assessment criteria on the tubal patency and diagnosis of uterine cavity lesions by transvaginal ultrasound real time three-dimensional hysterosalpingo-contrast-sonography(TVS RT-3D-HyCoSy).Material and MethodsMaterialPatients:528 infertile patients were chosen from our Assisted Reproduction and Gynecology Center from October 2013 to December 2014. The patients are with the ages from 19 to 46 years old. The average age is 29.5±4.7. The duration of infertility is 1 to 8 years. The average years is 3.2±2.9. Among them,227 cases are primary infertility (43.0%),301 cases are secondary infertility (57.0%). The inclusion criteria: exclude male infertility factors; no pelvic apparatus cancer found; no endocrine abnormalities observed; no systemic or heart, lungs, blood vessels and other vital organs diseases. Before hysterosalpingo, all the patients signed the informed consent. Hysterosalpingo was performed at the time of 3 to 7 days after the period.59 patients were observed by hysteroscopy after 1 to 3 months.Ultrasound examination instrument:GE Voluson E8 with CCI. Imaging probe is RIC5-9-D, center frequency 5.0～9.0 MHz, mechanical index 0.12～0.18.Pressure detection instrument:YLD-YZ-800U contrast agent injection device, testing pressure range is 0～90KPa, the maximum protection pressure set as 50KPa.Contrast agents:The positive contrast agent is SonoVue (Bracco company, Italian). A 59mg sulfur hexafluoride lyophilized powder mixes 5 ml 0.9% saline, and take the mixture of the whole 5 ml suspension liquid and 20 ml saline when carry out the operation. Negative contrast agent is 0.9% saline.Experimental animals:A total of 140 SPF KM mice provided by animal experiment center were used in these studies. All females are with the ages from 6 to 9 weeks old. The average age is (7±1) weeks. The weight is 29.1-38.5g, the average weight is (31.2±3.5) g, conventional farming in barrier system.Animals experimental soup:SonoVue (Bracco company,Italian), a 59mg sulfur hexafluoride lyophilized powder mixes 5 ml 0.9% saline, and take the mixture of the whole 5 ml suspension liquid and 15 ml saline when carry out the experiment. Xue ruixin(Hunan Kangrun Pharmaceuticals Co.,Ltd), a 3ml injection mixes 17ml 0.9% saline.MethodsExperiment 1:528 cases of infertile cases implement the positive combined with negative contrast agents TVS RT-3D-HyCoSy. After routine disinfection of uterine cavity, we inject 1.5～2.5ml saline with No.12 Foley’s tube into uterine cavity and block the internal opening of cervix. We take the conventional transvaginal scan to observe the position and the moving of uterus and ovaries to determine whether there is lesion in them, while observe the pelvic effusion, adhesion and calcifications, etc. The pressure detector is connected at one end of the fallopian tube filler tee. The ultrasound contrast agent injection device are connected at the other one end with the uterine cavity built-in pipe connection at the third end and set aside. Detailed steps are as follows:Start 3D mode key, adjust the maximum fan angle 179°, volume angle 120°, put the uterus and both ovaries in the maximum range of the volume of the box, and began 3D pre-scan. Making sure the volume of the box is located in the region of interest, we activate 4D key and start scanning. When the automatic dynamic storage time starts, opens the pressure detector and pushes positive contrast agents into uterine cavity continuously and slowly through the tee contrast agent injection port. According to records over time or dynamic imaging shows satisfactorily, the dynamic data set can be stored in the hard disk. We observe the situation and uniformity of contrast agent immediately with two dimensional ultrasonic contrasts. Then activate 3D key, collect 1-2 static 3D imaging data and store. After completion of HyCoSy, observed contrast agents surrounding around the bilateral ovaries and the dispersion evenness in pelvic cavity under the state of two-dimensional ultrasound imaging. Meanwhile, we use 3D volume acquisition to collect the distribution of the pelvis of contrast agent. Firstly, we record the amount of positive contrast agent injection and return flow, the pressure of pushing and pain degree of patients. Secondly, we apply the negative contrast agents to radiography. We reduce the liquid 0.5～1.0ml to cut the volume down and inject the negative contrast agent through catheter uniform with constant speed. Uterine cavity maintains a certain degree of expansion and stable pressure. We use two-dimensional ultrasound to scan the longitudinal and cross-sectional of coherent uterine to observe the uterine morphology, finishing degree of intimal surface and lesions. Thus we can record the location, size, shape, echo, and the relationship between endometrial internal blood supplies. Then we slightly increase pushing pressure to observe the flow from uterus to fallopian tube, the contrast agents overflow ovarian around and the fluid agglomeration or abnormal echo in the pelvic. We save the date in case of post-analysis. After operation, we will ask the patients to stay 20-30 minutes.After the HyCoSy completed, we recall the data of positive agent to observe the flow process of the contrast agent in the uterus and tubes pelvic. We analyze the tubal patency and the uterine cavity with the date of uterine tubal morphology, the contrast agent wrapping around ovarian and uterine, the contrast agent diffusion pelvic,contrast agent countercurrent as well as the amount of contrast agent push and return flow, bolus comprehensive analysis to assess the degree of pressure and pain in patients. Then we analyze the negative contrast agent image mainly to observe uterine cavity lesions and judge pathological change. Then observe intrauterine liquid flow direction and flow velocity, liquid flow in the fallopian tube, ovarian exists jet overflow, pelvic fluid surrounding the presence of an abnormal accumulation and belt-like hyperechoic, etc. By contrast with the hysteroscope, we analyze intrauterine lesions. Both diagnostic results consistent with the Kappa test, showed with sensitivity, specificity, positive predictive value, negative predictive value diagnostic results using χ2 difference test, with P<0.05 as statistically significant.Experiment 2:Taking the repetitive inspection to assess the tubal patency and uterine cavity lesions by the positive and negative contrast agent combined TVS RT 3D-HyCoSy. The same 528 cases of infertility patients, excluding removal of 13 cases of salpingectomy, image quality in 46 cases is poor.350 cases were selected with normative imaging data collection and consistent operation process. The two doctors in the 350 case, operator and the doctor who push the contrast agent are the same. Both of them are experienced high qualification doctor. After standardized training, certified by the same diagnosis standard after the HyCoSy and random sequence selected cases, two doctors, experienced high qualification doctor A and beginners of low qualification doctor B take independent evaluation through the HyCoSy images, injection contrast medium pressure, injection dose, regurgitation and degree of pain. For both the consistency of the diagnosis by chi-square test, with P< 0.05 as the difference was statistically significant.Experiment 3:Examine the safety of positive contrast agent to pelvic organs by injecting positive contrast agents into SPF kunming mice abdominal cavity.140 mice were assigned randomly into four groups, the experimental group A, the experimental group B and control group with 40 mice in each group, and 20 mice in blank group. Using the intraperitoneal injection, we injected 5ml SonoVue into each mouse in experimental group A,5ml Xue Ruixin in experimental group B and 5ml 0.9% N.S in control group. No processing in Blank group. All of them are conventional breaded in laboratory barrier facility.10 mice were killed randomly after injection 3 days,7 days,14 days and 21 days in each experimental and control groups, while 5 mice were killed each period of time in blank group. We observe the pelvic and record uterus fallopian tube shape, the surface smoothness and color, the surrounding tissue adhesion and pelvic effusion We cut the uterus, fallopian tubes, ovaries and surrounding connective tissue, which were placed in the volume fraction of 10% formaldehyde solution, HE staining and optical microscope observation. We observed whether the uterus and fallopian tube cavity expansion or adhesion, cavity wall structure level was clear, presence of inflammatory cells infiltration, uterus and fallopian tube hyperemia edema or not. We analyze statistical significant of each group.ResultsExperiment 1:A total of 1056 tubal in 528 cases of infertility, excluding tubal ectopic pregnancy surgery 13, so there are 1043 included in this study. Tubal patency results are divided into unobstructed, partially obstructed, obstructed.Positive contrast results:434 tubal unobstructed,368 tubes partially obstructed, 241 tubes obstructed. Among the obstructed tubes,129 proximal obstruction,112 middle and distal obstructed. There are 137 uterine with normal shape and smooth surface,36 uterine anomaly,5 submucosal fibroids,16 endometrial polyps,58 intrauterine adhesions and 12 uterine incision scar diverticulum.310 cases with no contrast agent counter-currented in myometrium and uterine venous plexus,151 cases with little amount of countercurrent and 68 cases with obvious countercurrent.Negative contrast results:There are 333 uterine with normal shape and smooth surface,36 uterine anomaly,4 submucosal fibroids,48 endometrial polyps,94 intrauterine adhesions and 13 uterine incision scar diverticulum.16 cases of pelvic adhesions were found after pelvic fluid increased and 4 cases of tubal fimbria parcel.The detection rates of intrauterine lesions by positive or negative contrast were 24.1%(127 cases) and 36.9%(195 cases). By Kappa coefficient test, bad consistency was showed in the results of positive contrast agents sonohysterography compared with the hysteroscopy (Kappa=0.087). Meanwhile, the negative contrast agents sonohysterography had well consistency (Kappa=0.781). There was statistically significant (P<0.05). Negative contrast agents Hysterography diagnosis of intrauterine lesions sensitivity, specificity, positive predictive value, negative predictive value were 98.1%.80.0%.98.1%.80.0%。Experiment 2:350 patients with 700 fallopian tubes were included in the study. The results of tubal patency degree assessed by two doctors are followed.327 tubals unobstructed,282 tubals partially obstructed,43 proximal obstructed and 48 middle and distal obstructed, which were assessed by doctor A.334 tubals unobstructed,276 tubals partially obstructed,43 proximal obstructed and 47 middle and distal obstructed, which were assessed by doctor B. Kappa coefficient test, both the diagnosis consistent (Kappa value= 0.977), there was no statistically significant difference (P>0.05). With regard to uterine malformation and intrauterine lesions,doctor A found 23 cases of uterine malformation,29 endometrial polyps,51 intrauterine adhesions and 8 scar diverticulum, while doctor B found 23 cases of uterine malformation,28 endometrial polyps,54 intrauterine adhesions and 7 scar diverticulum. Comparison between the discovery rates, there was no statistically significant difference (P> 0,05).Experiment 3:A total of 140 copies of the four groups of Kunming mice uterus, fallopian tubes and ovaries specimens, there were some specimens with varying degrees of inflammatory cell infiltration found by pathological examination showed. Among the blank group, the incidence of inflammation of 3-day、7-day、14-day、 21-day are 20%、0、0 and 40%.Among the experiment group A, the incidence of inflammation of 3-day、7-day、14-day、21-day are 10%、20%、0 and 20%, there was no significant difference between the groups. Among the experiment group B, the incidence of inflammation of 3-day、7-day、14-day、21-day are 20%、20%、10% and 0,there was no significant difference between the groups. Among the control group, the incidence of inflammation of 3-day、7-day、14-day、21-day are 30%、10%、0 and 20%, there was no significant difference between the groups. Differences in the results of the same period of the three groups were not statistically significant.ConclusionThe experiment 1:The positive and negative contrast agent combined application in TVS RT 3D-HyCoSy can accurately assess the degree of fallopian tubepatency and further improve the accuracy of the uterine cavity lesions. The positive contrast agent imaging can clearly show the shape of uterine cavity shape, the process and morphology of fallopian tubes in the pelvic cavity, the wrapping around the ovaries, pelvicdiffusion and contrast agent countercurrent. Combined with the contrast agent injection pressure, back flow the degree of pain, the comprehensive analysis can improve the accuracy of assessment of tubal patency and uterine malformation. However, the apparent contrast agents reflux would influences the observation of intrauterine form and lesions. But the intrauterine lesions, especially for smaller endometrial polyps and uterine adhesions can clearly show in the negative contrast agent, also some umbrella adhesions and pelvic adhesions can show. The positive and negative contrast agent combined application in TVS RT 3D-HyCoSy can provide valuable information for the clinical assessment of the overall situation of the reproductive tract.The experiment 2:Comparing the assessment of tubal patency, uterine malformations and intrauterine lesions discovery rate between the beginning junior doctor and experienced doctor in the positive and negative contrast agents combined TVS RT 3D-HyCoSy, there was no statistically significant, but the assessment of tubal passable beginner got low accuracy.The experiment 3:Two kinds of positive contrast agent are injected into the mice pelvis, Gross and light microscopy results showed that there was no statistically significant difference, and there was no statistically significant difference with the blank control group. There were inflammatory cells infiltration in some uterus appeared at all the four groups, but there was no statistically significant difference which indicating positive contrast agents used in ultrasound contrast hysterosalpingography was security. |
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