Preliminary quantitative and qualitative evaluation of contrast-enhanced ultrasonography (CEUS) in normal proliferative endometrium of infertile patients.

Endometrial receptivity plays a crucial role in the success of assisted reproductive technologies ( 1 ). The blood perfusion at the implantation site of the embryo is a key factor in assessing the endometrium’s capacity to accept the embryo ( 2 ). Adequate endometrial blood perfusion typically reflects a robust vascular network and favorable endometrial receptivity. Ultrasound has become the first-line method for screening and monitoring most gynecological conditions due to its distinct advantages. Traditional ultrasound evaluates endometrial receptivity based on uterine artery blood flow parameters, endometrial thickness, and blood perfusion, though it has notable limitations ( 3 , 4 ). Contrast-enhanced ultrasonography (CEUS) offers a real-time, dynamic assessment of microcirculatory blood flow in the endometrium and surrounding tissues ( 5 , 6 ). CEUS is increasingly applied in gynecology for evaluating space-occupying lesions such as endometrial lesions, uterine fibroids, adenomyosis, and gynecological tumors ( 7 ). Notably, CEUS offers higher diagnostic accuracy in distinguishing benign and malignant endometrial lesions compared to transvaginal ultrasound. As a novel ultrasound technique, CEUS is considered the optimal method for microvascular imaging. The most commonly used contrast agent, SonoVue, consists of microbubbles with 90% of the particles measuring less than 6 µm in diameter, which allows them to easily pass through capillaries, thereby enabling the visualization of microvascular perfusion in both normal uterine tissue and various focal uterine lesions ( 8 ). CEUS has shown significant promise in reproductive medicine ( 9 ). Numerous studies suggest that the detection of endometrial and subendometrial blood flow can be instrumental in assessing endometrial receptivity ( 10 , 11 ). However, a unified expert consensus on evaluating regional blood supply to the endometrium is lacking, and no established reference values are available. Most studies to date have focused on the ovulatory and implantation window periods. To avoid adverse pregnancy outcomes related to CEUS, this study uses endometrial pathological biopsy as the gold standard to investigate the imaging characteristics and reference ranges for quantitative parameters in different regions of interest within the normal proliferative endometrium of infertile patients, thereby providing reliable data to support the evaluation of endometrial receptivity. We present this article in accordance with the STARD reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2024-2737/rc ).

This retrospective diagnostic study included women of childbearing age in the proliferative phase of their menstrual cycle who visited our hospital for infertility treatment between April 2023 and July 2024. All participants underwent CEUS prior to hysteroscopy and endometrial pathological biopsy, following standard procedures. Most CEUS examinations were conducted between the 5th and 14th days of the menstrual cycle. Inclusion criteria included: (I) infertile patients with normal uterine morphology; (II) complete CEUS data; (III) endometrial morphology consistent with the proliferative phase on ultrasound (i.e., no visually discernible thinning; (IV) absence of endometrial, fallopian tube, or ovarian lesions; (V) normal endocrine hormone tests. Exclusion criteria were as follows: (I) patients with abnormal proliferative endometrium based on pathological findings, including complex endometrial hyperplasia, atypical endometrial hyperplasia, endometrial cancer, or chronic endometritis; (II) patients who had used medication affecting blood supply within three months prior to the examination; (III) patients with allergies to SonoVue contrast agent components; (IV) patients with a history of malignant tumors or blood system-related disorders; (V) patients with poor compliance or difficulty completing the study; (VI) patients with poor quality of contrast video. The term “normal proliferative endometrium” in this context specifically refers to histopathologically verified endometrium exhibiting typical morphological and histological characteristics of the proliferative phase. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (No. LLSY-2020-055) and informed consent was obtained from all individual participants. The GE LOGIQ E9 color ultrasound diagnostic apparatus was used, equipped with an IC 5–9 probe (frequency: 8.0 MHz, mechanical index: 0.8–1.2). The ultrasound contrast agent, consisting of sulfur hexafluoride microbubbles (SonoVue ® ; Bracco Suisse SA, Switzerland), was prepared as a suspension with 2.4 mL of contrast agent mixed with 10 mL of normal saline. Transvaginal routine two-dimensional ultrasound and color Doppler ultrasound examination: prior to the transvaginal ultrasound examination, the patient was instructed to empty the bladder and assume a lithotomy position. The thickness of the endometrium was measured in the mid-sagittal plane using two-dimensional ultrasound, followed by a color Doppler ultrasound to record the blood flow parameters of both uterine arteries, including peak systolic velocity (Vmax), minimum diastolic velocity (Vmin), resistance index (RI), pulsatility index (PI), and other hemodynamic parameters. CEUS examination: following the completion of the routine ultrasound scan, the machine was adjusted to contrast mode. During the examination, the subjects were instructed to breathe slowly and evenly to ensure image stability. The video acquisition system was then activated to capture and record the entire process of endometrial angiography and perfusion phase dynamics. At least 2 minutes of dynamic imaging were saved and stored on the workstation. Time-intensity curve (TIC) analysis: TICs for regions of interest (ROIs) were automatically generated by the ultrasound instrument’s built-in post-processing software. Two or three ROIs were selected in the endometrial region, two ROIs in the subendometrial region (defined as within 2 mm beyond the outer edge of the endometrium), and two ROIs in the myometrium along the same vertical-horizontal line. All ROIs were set to dimensions of 10 mm × 1 mm and 2 mm × 2 mm. The standardized TIC was derived using the wash-in formula. Quantitative indices were calculated for each ROI, and the mean values of the two ROIs from each region (endometrium, subendometrium, and myometrium) were used for analysis. The quantitative indices calculated for the endometrial, subendometrial, and myometrial regions included peak intensity (Pi), ascending branch slope (K), area under the curve (AUC), and time to peak (TtoPK). All transvaginal two-dimensional ultrasound, color Doppler, CEUS, and TIC analyses were performed by a single physician with more than three years of experience in gynecological ultrasound and CEUS, following standardized training. Clinical data and relevant medical history: this included the patient’s time of pregnancy preparation, menstrual history, pregnancy and childbirth history, history of intrauterine procedures, and any relevant past medical history. Two-dimensional and color Doppler ultrasound hemodynamic parameters: the endometrial thickness and type were classified according to Gonen’s classification standard ( 12 ): Type A endometrium: characterized by a three-line appearance, with strong echogenicity in both the outer layer and the center, and a low-echo or dark area between the outer layer and the midline of the uterine cavity. Type B endometrium: displays uniform medium-intensity echogenicity, with intermittent or unclear midline echoes. Type C endometrium: homogeneous with strong echoes and no midline echo within the uterine cavity. Endometrial echo homogeneity was classified as homogeneous (uniform and anteroposteriorly symmetrical endometrium) or heterogeneous (asymmetry or cystic endometrial appearance) according to the IETA consensus ( 13 ). Applebaum classification ( 14 ) was used to categorize the endometrial and subendometrial blood flow as: Type I: blood vessels pass through the outer hypoechoic band of the endometrium but do not reach the outer edge of the hyperechoic endometrium. Type II: Blood vessels pass through the outer edge of the hyperechoic endometrium but do not enter the hypoechoic area. Type III: blood vessels penetrate into the hypoechoic area of the endometrium. The following hemodynamic parameters were recorded: Vmax, Vmin, PI, and RI. CEUS-related indicators: CEUS qualitative analysis indicators: (a) enhancement intensity of the ROI, categorized as high, equal, or low enhancement; (b) boundary definition: whether the boundary of the ROI was clear; (c) contrast agent distribution within the ROI, classified as uniform enhancement, sub-uniform enhancement, or non-uniform enhancement. According to the literature, uniform enhancement is defined as nearly complete perfusion of the endometrial area, sub-uniform enhancement as abnormal low perfusion in less than 1/3 of the endometrial area, and non-uniform enhancement as abnormal low perfusion in more than 1/3 of the endometrial area. CEUS quantitative analysis indicators: Pi, K, AUC, and TtoPK. Data analysis was performed using SPSS 25.0 (IBM, Armonk, NY, USA). Normally distributed continuous data are presented as mean ± standard deviation (SD), with comparisons between two groups performed using the independent sample t -test. One-way analysis of variance (ANOVA) with the least significant difference (LSD) test was used for comparisons between three or more groups, and the LSD t -test was applied for multiple comparisons between two groups. Non-normally distributed data are presented as median (lower quartile, upper quartile), and comparisons were made using the Kruskal-Wallis H test. Categorical and ranked data are expressed as constituent ratios (%) or rates (%), and statistical analysis was performed using the Kruskal-Wallis H test. The intraclass correlation coefficient (ICC) was used to assess the consistency between the two methods. A P value of <0.05 was considered statistically significant.

In this study, clinical data and CEUS examination results were collected for 60 infertile patients. The study flowchart is presented in Figure S1 . Fifteen patients were excluded due to the presence of chronic endometritis in endometrial biopsy results, and five were excluded due to poor-quality angiography videos. Ultimately, 40 patients with normal proliferative endometrium were included. The mean age of the patients was 34.59±4.50 years, and the duration of infertility ranged from 22.51 to 38.22 months. Among the participants, 28 had a history of uterine cavity surgery, 22 had a history of abortion, 3 had a history of ectopic pregnancy, and 23 had irregular menstruation. All 37 patients underwent CEUS examinations between the 5th and 14th days of the menstrual cycle, while the remaining 3 patients were examined on the 15th, 16th, and 19th days due to a longer menstrual cycle. The endometrial thickness of one patient with a normal proliferative endometrium was not measurable due to uterine effusion and separation of the uterine cavity line. The mean endometrial thickness of the remaining 39 patients was 5.84±2.23 mm, with 6 cases of Type A endometrium, 29 cases of Type B endometrium, and 4 cases of Type C endometrium. Of the 39 patients, 33 exhibited uniform endometrial echoes on two-dimensional ultrasound, while 7 showed uneven endometrial echoes. A total of 25 patients with normal proliferative endometrium underwent Applebaum classification, with 5 cases of Type I blood flow, 8 cases of Type II blood flow, and 12 cases of Type III blood flow. Four patients were excluded from analysis due to large spectral fluctuations, defined as the inability to acquire ≥3 consecutive stable cardiac cycles, due to patient anxiety during color Doppler ultrasound (manifested by involuntary pelvic muscle contractions) impairing uterine artery Doppler signal acquisition. A total of 36 patients underwent color Doppler ultrasound, with the following mean values recorded: Vmax 42.52 (35.90, 56.74) cm/s, Vmin 7.3 (4.52, 10.55) cm/s, PI: 2.08±0.51, RI: 0.82±0.07. The CEUS images of normal proliferative endometrium predominantly exhibited homogeneous enhancement, with clear delineation between the myometrium and subendometrial region. The enhancement intensity was primarily categorized as low enhancement (22/40) and equal enhancement (14/40) ( Figure 1A,1B ). Contrast-enhanced ultrasonography. (A) A CEUS image of a 31-year-old patient with normal proliferative endometrial infertility, with no history of obstetric or gynecological diseases or uterine cavity manipulation. The boundary between the endometrium and subendometrial region is clearly visible (indicated by the white arrows). The endometrial region is demarcated from the myometrium, with homogeneous and equal enhancement (indicated by the blue arrow), and the enhancement intensity of the myometrium is similar to that of the endometrium (indicated by the black arrow). (B) A CEUS image of a 28-year-old patient with normal proliferative endometrial infertility and a history of one miscarriage. The boundary between the endometrium and subendometrium is clearly visible (indicated by the white arrows). The endometrial region is clearly demarcated from the myometrium, with uniform low enhancement in the endometrium (indicated by the blue arrow), while the myometrial enhancement intensity is greater than that of the endometrium (indicated by the black arrow). (C) TICs for seven ROIs, with 10 mm × 1 mm ROIs placed in various regions of the endometrium (yellow, gray, blue), subendometrium (red, green), and myometrium (orange, purple). (D) Time-intensity curves for six ROIs, with 2 mm × 2 mm ROIs placed in the middle and upper parts of the anterior and posterior walls of the endometrium (blue, yellow), subendometrium (green, red), and myometrium (purple, orange). The quantitative indices calculated by the software included Pi, K, AUC, and TtoPK. A(Pi), peak intensity; ATm, arrival time; AUC, area under the curve; B, basic intensity; CEUS, contrast-enhanced ultrasonography; Grad, mean gradient for the peak intensity; K, ascending slope; MSE, squared error; Pi, peak intensity; ROI, region of interest; TIC, time-intensity curve; TtoPK, time to peak. The morphology of the TIC in the endometrial region, subendometrial region, and myometrium was similar. In the endometrial region, the TIC initially displayed a small segment of baseline signal, gradually increasing to Pi, before slowly decreasing to the plateau phase. In contrast, both the subendometrial region and myometrium showed a rapid initial enhancement, peaking at a higher intensity than the endometrial region, followed by a gradual decrease to the plateau phase, with a more pronounced decline compared to the endometrial region ( Figure 1C,1D ). The 10 mm × 1 mm ROI was designated as ROI group 1, and the 2 mm × 2 mm ROI was designated as ROI group 2. Relevant quantitative parameters, including Pi, K, AUC, and TtoPK, were obtained for both groups ( Table 1 ). In ROI group 1, the Pi of the endometrial region was 16.19±4.28 dB, K was 0.24±0.09, AUC was 1,257.95±301.83 dB, and TtoPK was 18.29±4.75 s. For ROI group 2, the Pi of the endometrial region was 17.75±4.95 dB, K was 0.26±0.08, AUC was 1,302.21±317.51 dB, and TtoPK was 16.47 (13.92, 20.41) s. Consistency between the 10 mm × 1 mm and 2 mm × 2 mm measurements for different ROIs was good ( Table 1 ). Data are presented as mean ± standard deviation or median (interquartile range). ROI group 1, 10 mm × 1 mm ROI ; ROI group 2, 2 mm × 2 mm ROI. AUC, area under the curve; CEUS, contrast-enhanced ultrasonography; ICC, intraclass correlation coefficient; K, ascending slope; Pi, peak intensity; ROI, region of interest; TtoPK, time to peak. Statistical analysis revealed significant differences in Pi and K values between the endometrial and myometrial regions in different ROIs (P0.05). Comparison of the CEUS quantitative indicators among different regions of interest within ROI group 1 showed statistically significant differences in Pi, K, and AUC. Further pairwise comparisons indicated that the Pi in the endometrial region was lower than that in the myometrium, and both K and AUC in the endometrial region were significantly lower than those in the subendometrial and myometrial regions, with the myometrium demonstrating higher values than the subendometrium (P0.05). Similarly, in ROI group 2, significant differences in Pi, K, and AUC were observed between the three regions of interest. Further pairwise comparisons showed that Pi and K values in the endometrial region were lower than those in the myometrial region, and AUC in the endometrial region was significantly lower than in the subendometrial and myometrial regions, with the myometrium showing higher values than the subendometrium (P0.05) ( Table 2 ). Data are presented as mean ± standard deviation or median (interquartile range). ROI group 1, 10 mm × 1 mm ROI ; ROI group 2, 2 mm × 2 mm ROI. AUC, area under the curve; CEUS, contrast-enhanced ultrasonography; K, ascending slope; Pi, peak intensity; ROI, region of interest; TtoPK, time to peak. When comparing the CEUS quantitative indicators among different Applebaum classifications under color Doppler ultrasonography, no statistically significant differences in Pi, K, TtoPK, or AUC were found among the three regions of interest (P>0.05) ( Table 3 ). Data are presented as mean ± standard deviation. AUC, area under the curve; CEUS, contrast-enhanced ultrasonography; K, ascending slope; Pi, peak intensity; TtoPK, time to peak.

A healthy endometrium is crucial for successful pregnancy, as approximately two-thirds of pregnancy failures are attributable to impaired endometrial receptivity ( 15 ). Endometrial angiogenesis plays a pivotal role in embryo implantation ( 16 ), and histological studies have demonstrated that both the endometrial and subendometrial regions are essential for endometrial receptivity ( 17 , 18 ). Several methods are available for evaluating endometrial receptivity, including biomarker assays, genetic markers, and ultrasonography. While endometrial microvascular density is considered the gold standard for evaluation, its invasive nature limits its clinical applicability. Ultrasound is a safe, non-invasive, and reproducible modality that can assess uterine perfusion and the supply of uterine arteries through indicators such as blood flow status and velocity within the endometrium ( 19 , 20 ). However, as a significant proportion of the blood flow from the uterine artery is directed to the myometrium, using the uterine artery RI to reflect endometrial perfusion has certain limitations ( 20 ). The terminal branches of the uterine artery, which supply blood to the endometrium, make it more accurate to evaluate endometrial perfusion and surrounding tissue. However, due to the small diameter and low flow velocity of the spiral arteries, capturing their blood flow signals with conventional color Doppler ultrasound remains challenging. CEUS is a non-invasive imaging technique that allows for continuous, real-time observation of micro-perfusion within tissues using contrast agents and nonlinear imaging technology. CEUS enables effective qualitative analysis by assessing the enhancement sequence, intensity, and potential filling defects, offering greater sensitivity than traditional color Doppler ultrasound. When combined with the TIC, CEUS can intuitively and quantitatively evaluate microcirculation and microvascular conditions, providing valuable information for clinical diagnosis ( 21 ). Currently, SonoVue, a contrast agent composed of microbubbles containing sulfur hexafluoride gas, is the most widely used contrast agent both domestically and internationally. SonoVue is highly stable, acts as a pure blood pool contrast agent, and has high safety, as it enters only the microvessels and does not permeate tissues. Given the thinness of the endometrium during menstruation and the continuous shedding and bleeding that occurs, it is difficult to accurately assess endometrial blood perfusion using CEUS. Although there is no experimental evidence to suggest that the use of ultrasound contrast agents during the ovulatory period adversely affects pregnancy or subsequent outcomes, this study focused on the normal proliferative endometrium, investigating the imaging characteristics and quantitative parameter ranges of different ROIs. Future research could extend to studies conducted during different menstrual cycles. Moreover, some studies have suggested that the location and size of the ROI can influence the accuracy and reliability of the diagnosis. Therefore, selecting appropriate ROIs is crucial for the quantitative analysis of endometrial CEUS. At present, there is no consensus among experts regarding the optimal size and positioning of ROIs. In this study, the authors referred to previous literature on ROI positioning, integrating clinical experience and prior research conducted by the team. Two ROIs were chosen for analysis: 2 mm × 2 mm and 10 mm × 1 mm, which were intended to capture subtle blood flow and broader regional blood flow, respectively. The results demonstrated no significant differences in the ultrasound contrast indices between the two ROIs, and their consistency was high. Both ROIs were found to be effective in reflecting endometrial blood perfusion. This study utilized TIC to quantitatively assess blood perfusion in the endometrial and subendometrial regions, whereas previous studies on endometrial and subendometrial blood flow have predominantly used the Applebaum classification with two-dimensional color Doppler ultrasound. Research has shown that type III blood flow is more favorable for successful pregnancy ( 22 , 23 ). However, this assessment method is highly dependent on the operator’s skill and the ultrasound equipment. In the present study, quantitative parameter analysis was conducted on patients categorized according to the Applebaum classification using color Doppler. The results revealed no significant differences in quantitative parameters among the three Applebaum types, suggesting that using color Doppler ultrasound for Applebaum classification to assess blood flow in the endometrium and subendometrium may have limitations. In contrast, CEUS can more objectively display the status of endometrial and subendometrial blood flow, demonstrating extremely high sensitivity to changes in blood perfusion. In terms of the morphology of the TIC, the TICs for the endometrial region, subendometrial region, and myometrium exhibit similarities; however, the TICs of the endometrial region predominantly display slow-rising and slow-falling patterns, with rounded peaks and smooth waveforms. In contrast, the curves of the subendometrial region generally exhibit a more rapid rise and fall compared to the endometrial region, with a slightly rounded peak. The TICs of the myometrium, on the other hand, typically show a relatively rapid rise and fall, with a small, sharp peak waveform. Regarding the quantitative parameters of the TICs, the results of this study indicated that the normal proliferative endometrium in infertile patients demonstrated uniform low or equal echo levels on CEUS, with both peak time and Pi lower than those observed in the subendometrial region and myometrium. These findings are consistent with the anatomical characteristics of the uterine artery branches ( 7 , 24 ). The enhancement of the endometrium in normal uterine CEUS initiates from the periphery and progresses centripetally. The spiral artery, which has a small diameter and predominantly supplies blood to the endometrium, results in the endometrium being relatively deficient in blood supply compared to the myometrium. Consequently, the contrast perfusion in the endometrium occurs more slowly than in the myometrium, and its enhancement is also less pronounced. These observations align with the results of this study. By analyzing the normal ultrasound contrast behavior of the endometrium, subendometrial region, and myometrium, and establishing the corresponding ranges for quantitative parameters, this study provides valuable insights into uterine blood perfusion and endometrial function for clinical applications. However, there are several limitations in this study. First, the sample size of this study was relatively small, which may have limited the impact of other potential variables. Second, it was conducted at a single center, which may restrict the generalizability of the findings. Furthermore, the lack of a control group consisting of women with proven fertility is a notable limitation. The inclusion of such a group would have allowed for a direct comparison of CEUS parameters, facilitating the establishment of robust diagnostic or prognostic criteria. Additionally, since the study subjects were exclusively infertile patients with a histopathologically confirmed normal proliferative endometrium, the ultrasound parameters could not be compared across different uterine pathological states. Therefore, future large-scale, multicenter studies that include a fertile control group are required to validate and extend our findings.

This study investigated the imaging characteristics and reference values of quantitative parameters derived from CEUS in the normal proliferative endometrium of infertile patients. The combination of ultrasound contrast imaging with transvaginal ultrasound may provide a convenient, useful, and non-invasive method for assessing endometrial receptivity.

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