Abstract
Background
Prospective clinical research on the use of ultrasound contrast agents for tubal patency assessment in hysterosalpingo-contrast sonography (HyCoSy) is limited. This study sought to evaluate the efficacy and safety of perfluoropropane human albumin microspheres as an ultrasound contrast agent for HyCoSy in assessing fallopian tube patency, using laparoscopy (LSC) as the gold standard.
Methods
This prospective, double-blind, phase III study was conducted at 11 centers in China from April 2022 to April 2023 (ClinicalTrials.gov: CTR20220578). Infertile women aged between 18 and 45 years, who were scheduled for tubal patency testing, were eligible to participate in the study. The HyCoSy examination was performed by an experienced sonographer, and the image analysis was conducted by two experienced sonographers. Subsequently, within one menstrual cycle, LSC was performed by gynecologists who were blinded to the HyCoSy results. The accuracy of HyCoSy was calculated using the LSC results as the gold standard. In addition, a binary logistic regression model was applied to analyze the clinical and ultrasound factors associated with false-positive and false-negative results. Safety assessments were conducted before HyCoSy, within one week after HyCoSy, and before discharge from hospital following LSC.
Results
A total of 120 participants were enrolled in the study, of whom 112 completed both HyCoSy and LSC. Two participants who had previously undergone unilateral salpingectomy, and one participant whose fallopian tube evaluation result was recorded as “uncertain” on one side by HyCoSy, resulting in a total of 221 fallopian tubes included in the efficacy analysis. Compared to LSC, the overall accuracy of HyCoSy was 88.24% (195/221). The total false-positive rate and false-negative rate were 3.62% (8/221) and 8.14% (18/221), respectively. The false-positive group had a higher proportion of previous ectopic pregnancy and a higher proportion of ovaries adjacent to the uterus (P=0.03, P=0.01), while the false-negative group had a higher incidence of contrast intravasation (P=0.046). In the safety assessment, pain and contrast intravasation were common treatment-emergent adverse events.
Conclusions
Perfluoropropane human albumin microspheres can be used to effectively evaluate tubal patency through HyCoSy, and their diagnostic accuracy is good compared to the gold standard, LSC. Additionally, this contrast agent was associated with fewer and milder adverse reactions, making it suitable for clinical application.
Keywords: Hysterosalpingo-contrast sonography (HyCoSy), contrast agents, tubal patency, diagnostic accuracy, double-blind
Introduction
Tubal disease is a major cause of female infertility, accounting for 30–35% in female infertility cases (1). Thus, tubal patency testing is a crucial first step in infertility evaluation (2,3). The main clinical assessment methods include laparoscopy (LSC) with the chromotubation dye test, X-ray hysterosalpingography, and hysterosalpingo-contrast sonography (HyCoSy).
LSC with the chromotubation dye test remains the gold standard for evaluating tubal patency; however, it is invasive, costly, and associated with anesthesia-related side effects (4,5). X-ray hysterosalpingography is one of the main procedures used to assess tubal patency and remains the first-line diagnostic examination at some fertility centers; however, it involves exposure to ionizing radiation and the use of potentially allergenic contrast media (5). Following recent advances in ultrasound contrast agents and ultrasound imaging technology, HyCoSy is increasingly being used as a preliminary diagnostic examination method due to its safety, good patient tolerance, high diagnostic accuracy, lack of radiation, and procedural simplicity (6-8).
Due to the stability of second-generation ultrasound contrast agents—microbubbles composed of inert gases encapsulated in flexible shells such as phospholipids or albumin—HyCoSy can now be used to monitor the flow of contrast agents in the fallopian tubes in real time, clearly identifying the specific location and patency of fallopian tube obstructions (9). However, currently available agents such as SonoVue, Sonazoid, Optison, and Definity have yet to receive regulatory approval for HyCoSy applications specifically.
Perfluoropropane human albumin microspheres (Lidaxing™, Lizhuo Pharmaceutical Co., Ltd., Xiamen, China) are a freeze-dried injectable contrast agent, consisting of gas-filled microspheres encapsulated in solid shells of heat-denatured human albumin. Complete phase I/II/III clinical data have shown the safety of this contrast agent in human clinical applications (10). It has been approved for left ventricular contrast-enhanced ultrasound in China, and some clinical studies suggest that it is safe for use in HyCoSy (11,12).
Therefore, the main purpose of this study was to evaluate the efficacy and safety of perfluoropropane human albumin microspheres in HyCoSy for assessing fallopian tube patency using LSC as the gold standard. To the best of our knowledge, this was the first double-blind, multicenter, prospective phase III study to evaluate the utility of this ultrasound contrast agent in HyCoSy for assessing fallopian tube patency. We present this article in accordance with the STARD reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1506/rc).
Methods
This prospective, double-blind, phase III study was conducted at 11 centers in China from April 2022 to April 2023 (ClinicalTrials.gov: CTR20220578), and was approved by independent ethics committees or independent review boards of all 11 participating clinical research centers in accordance with national and local regulations. Written informed consent was obtained from all the participants. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.
Participant selection
Infertile women aged 18–45 years at each center, who were required to undergo a fallopian tube patency test and met the criteria for LSC, were recruited for the study. Before enrollment, all participants underwent a standard infertility workup, which typically included ovulation assessment (e.g., basal body temperature charting and mid-luteal progesterone level testing), partner semen analysis, and a baseline gynecological ultrasound to assess uterine morphology and ovarian reserve, to ensure that tubal factors were a suspected cause of infertility, justifying the need for tubal patency testing.
The exclusion criteria were as follows: (I) a history of clinically severe systemic diseases or an inability to tolerate surgery; (II) mental illness or epilepsy; (III) coagulation disorders or ongoing therapy with anticoagulant drugs; (IV) abdominal hernia, diaphragmatic hernia, or diffuse peritonitis with intestinal obstruction; (V) malignant lesions of the cervix or uterine cavity; (VI) suspected malignant or space occupying lesions, or large masses (>10 cm) in the pelvic cavity; (VII) acute inflammation of the internal or external reproductive organs; (VIII) a positive blood pregnancy test result; (IX) known allergies to narcotic, atropine, albumin, or other blood products; (X) participation in other drug clinical trials within 3 months prior to screening; (XI) a recent blood donation or a history of significant blood loss within 3 months prior to screening; (XII) human immunodeficiency virus or syphilis positive; and/or (XIII) any other condition deemed unsuitable for participation by the investigators.
Sample size
According to the literature, the accuracy of HyCoSy compared to the gold standard LSC is approximately 85–94% (13-15). Assuming an expected accuracy of 90%, a sample size of 83 participants was required to ensure 80% power and a 5% type I error rate. Considering an estimated dropout rate, the total sample size was set at 120, which was expected to provide sufficient validity data for this study.
Contrast agent and contrast medium preparation
Perfluoropropane human albumin microspheres (Lidaxing™, Lizhuo Pharmaceutical Co., Ltd., Xiamen, China) were chosen as the contrast agent. The contrast agent was reconstituted and diluted with 0.9% sodium chloride injection to a final volume of 20 mL for later use. The detailed contrast medium preparation steps are described in the Appendix 1.
Laboratory examinations
Laboratory tests, including complete blood count, coagulation function, blood biochemistry, blood pregnancy, and vaginal secretion tests, were conducted within one week prior to HyCoSy. Immunological, electrocardiogram, and gynecological ultrasound examinations were conducted within three months before HyCoSy. In addition, complete blood count, blood biochemistry, and electrocardiogram examinations were re-conducted after HyCoSy and LSC.
Performance of HyCoSy and image evaluation
All the ultrasound machines in the study (Voluson E8 and E10 Expert, GE Healthcare, Zipf, Austria; Nuewa R9, Mindray, Shenzhen, China) were equipped with a transvaginal three-dimensional volume ultrasound probe and low mechanical index contrast-enhanced ultrasound specific imaging technology. HyCoSy was performed within 3–7 days after menstruation, and sexual activity was prohibited for the three days preceding HyCoSy. For details of the HyCoSy operation steps, see Appendix 1.
Based on contrast agent visualization, a diagnosis of tubal patency was made when the contrast agent filwed naturally and clearly within the fallopian tube, and flowed freely from the fimbriated ends of the fallopian tubes, and a strong echogenic ring appeared around the ovaries (Figure 1A). Conversely, a diagnosis of tubal obstruction was made if the entire fallopian tube was not visible, if the middle and distal ends were not visible, if the distal end was enlarged, there was no contrast agent flowed from the fimbriae, and no strong echogenic ring appeared around the ovary (Figure 1B). If the fallopian tube evaluation was inconclusive, the reason was documented, and the result was categorized as “uncertain”; these cases were excluded from the diagnostic efficacy analysis.
Figure 1.
HyCoSy imaging examples of tubal patency and obstruction. (A) HyCoSy showing fully visualized bilateral fallopian tubes with contrast agent flowing freely from the fimbriae (arrows), indicating tubal patency. (B) HyCoSy showing non- visualization of the entire right fallopian tube (short arrow), and the middle and distal ends of the left fallopian tube (long arrow), indicating obstruction. HyCoSy, hysterosalpingo-contrast sonography; L, left; R, right.
Prior to study commencement, all sonographers underwent standardized training on image acquisition protocols and interpretation criteria to ensure consistency. The HyCoSy procedure and image analyses were performed by an experienced ultrasonographer with more than 5 years of experience in HyCoSy, and the results were reviewed by another experienced sonographer. The evaluation results were recorded after consensus was reached.
LSC evaluation of fallopian tube patency
After HyCoSy, the participants who were willing to undergo LSC were scheduled for LSC with the chromotubation dye test, ensuring a maximum interval of one menstrual cycle between the two examinations. During LSC, tubal patency was assessed first, followed by any necessary therapeutic procedures such as the management of pelvic adhesions or fibroids. If methylene blue was observed at the end of the fimbriae, the fallopian tube was considered patent (Figure 2A), otherwise it was considered obstructed (Figure 2B). Again, if the fallopian tube evaluation was inconclusive, the reason was documented, and the result was recorded as “uncertain”; these cases were excluded from the diagnostic efficacy analysis.
Figure 2.
LSC with the chromotubation dye test corresponding to Figure 1. (A) LSC with the chromotubation dye test showing methylene blue fluid flowing freely from both fimbriae (arrows), confirming tubal patency. (B) LSC with the chromotubation dye test showing no filling of methylene blue fluid in the entire right fallopian tube, and no methylene blue fluid flowing freely from the fimbriae (short arrow); methylene blue liquid accumulation can be seen in the middle and distal ends of the left fallopian tube, with no methylene blue fluid flowing freely from the fimbriae (long arrow), confirming obstruction. LSC, laparoscopy; L, left; R, right.
The evaluation of LSC fallopian tube patency was performed by a professor of obstetrics and gynecology with 10 years of clinical experience, who was blinded to the HyCoSy results during the assessment. To further validate the accuracy and consistency of the LSC results, the LSC videos were archived and independently reviewed by another professor.
The LSC diagnostic results served as the gold standard for assessing fallopian tube patency. A false-positive result was defined as HyCoSy indicating tubal obstruction when LSC indicated tubal patency. Conversely, a false-negative result was defined as HyCoSy indicating patency when LSC indicated obstruction.
Safety evaluation
Safety assessments were conducted before HyCoSy, within one week after HyCoSy, and before discharge following LSC. The evaluated variables included the symptoms and clinical signs observed during the study (e.g., pain, nausea and vomiting, pelvic infections, and contrast intravasation), laboratory indicators (e.g., hematology and biochemistry), and electrocardiogram results.
Treatment-emergent adverse events (TEAEs) were defined as any unfavorable or unintended sign, symptom, or disease that occurred on or after the administration of the investigational contrast agent, and before the completion of the safety follow-up period. Any adverse event (AE) present before contrast agent administration was only considered a TEAE if its severity worsened after administration.
A pre-defined three-tier classification system was used to grade the severity of all AEs based on their impact on the participants’ daily life as follows: mild (Grade 1): easily tolerated, causing minimal discomfort, and not interfering with normal daily activities; moderate (Grade 2): causing sufficient discomfort to interfere with or limit normal daily activities; and severe (Grade 3): preventing or significantly hampering normal daily activities.
Pain scores were obtained using a Visual Analog Scale (VAS) after HyCoSy. Patients indicated the maximum pain intensity they experienced by making a vertical line on the VAS. Under the VAS, a score as 0 indicated “no” pelvic pain, a score of 1–4 indicated “mild” pelvic pain, a score of 5–7 indicated “moderate” pelvic pain, and a score of 8–10 indicated “severe” pelvic pain. All AEs, serious AEs, changes in vital signs, clinical laboratory variables, and physical examination findings were recorded and assessed.
Blinding
In this study, both the patients and LSC doctors were blinded to the HyCoSy results. To ensure that the clinical trial adhered to standardized blinding methods and produced authentic and reliable data, HyCoSy and LSC were performed independently by researchers from the Department of Ultrasound and the Department of Gynecology, respectively. The two departments were independent, with no overlapping personnel. After each HyCoSy, the results were securely stored in a local database, and were inaccessible to the gynecologists. The HyCoSy results were not disclosed to the gynecologists until after LSC completion, ensuring strict blinding throughout the study.
Statistical analysis
The data were analyzed using SAS® version 9.4 (SAS Inc., Cary, NC, USA). The continuous data are expressed as the mean ± standard deviation, and the rank data are expressed as the frequency (n) and percentage. The primary diagnostic performance metrics (e.g., accuracy, sensitivity, and specificity) for HyCoSy were calculated with the fallopian tube as the statistical unit of analysis. The accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR–), and 95% confidence interval (CI) of HyCoSy in evaluating tubal patency were calculated, with the LSC results as the gold standard. The inter-observer agreement between the two sonographers for HyCoSy image interpretation was assessed using Cohen’s kappa (κ) statistic. A binary logistic regression model was applied to analyze the clinical and ultrasound factors associated with false-positive and false-negative results. P values <0.05 were considered significant.
Results
Participant characteristics
During the 12-month study period, 141 participants were screened from 11 centers in China, of whom 21 were excluded due to non-compliance with the inclusion criteria. Thus, ultimately, 120 participants were enrolled in the study. Among them, 117 participants underwent HyCoSy, and 116 participants (99.15%, 116/117) successfully completed HyCoSy. All the participants who underwent HyCoSy received only one injection of contrast agent, with an average injection of 17.0±4.96 mL (range, 3–20 mL). Four participants withdrew from the study after HyCoSy, resulting in 112 participants successfully completing LSC and being included in the efficacy evaluation. A flowchart showing the inclusion and exclusion of the study participants is presented in Figure 3.
Figure 3.
Flowchart of participants enrolled in the study and included in the analysis. HyCoSy, hysterosalpingo-contrast sonography; LSC, laparoscopy.
The median time between HyCoSy and LSC was 1 day (range, 0–60 days), and 110 participants (98.2%) had an interval of 6 days or less. The remaining two participants completed both procedures at intervals of 32 and 60 days, respectively, due to irregular menstrual cycles, which was in accordance with the research protocol. Table 1 summarizes the characteristics of the participants who underwent HyCoSy and LSC.
Table 1. Demographic characteristics of HyCoSy and LSC participants.
| Characteristic | HyCoSy (n=117) | LSC (n=112) |
|---|---|---|
| Age (years) | 31.07±4.34 | 30.90±4.26 |
| 18–34 | 94 (80.34) | 93 (83.04) |
| 35–45 | 23 (19.66) | 19 (16.96) |
| Ethnic | ||
| Han nationality | 112 (95.73) | 108 (96.43) |
| Other | 5† (4.27) | 4‡ (3.57) |
| Height (cm) | 158.54±5.14 | 158.54±5.21 |
| Weight (kg) | 56.44±9.19 | 56.41±9.37 |
| Infertility duration (years) | 3.22±2.81 | 3.27±2.83 |
| Infertile type | ||
| Primary | 65 (55.56) | 63 (56.25) |
| Secondary | 52 (44.44) | 49 (43.75) |
Data are expressed as mean ± standard deviation or n (%). †, other ethnic groups included one case each of Hui, Miao, and Tujia, and two cases of Zhuang. ‡, other ethnic groups included one case each of Hui, Miao, Tujia, and Zhuang. HyCoSy, Hysterosalpingo-Contrast Sonography; LSC, laparoscopy.
HyCoSy and LSC diagnosis results
Among the 112 participants who completed both HyCoSy and LSC, two underwent unilateral salpingectomy, resulting in a total of 222 fallopian tubes available for analysis. According to the HyCoSy results, 134 fallopian tubes were classified as patent, 87 as occluded, and one as “uncertain”. Conversely, according to the LSC results, 125 fallopian tubes were classified as patent and 97 as occluded (Table 2). The data of the patient whose fallopian tube evaluation result was recorded as “uncertain” on HyCoSy was excluded from the diagnostic efficacy analysis. Therefore, a total of 221 fallopian tubes from 112 participants were included in the efficacy analysis. The overall diagnostic accuracy of HyCoSywas 88.24% (sensitivity 81.44%, specificity 93.55%, PPV 90.80%, NPV 86.57%, LR+ 12.62, LR– 0.20, and Kappa 0.76). The 95% confidence lower limit was 83.99%; thus, the primary efficacy endpoint of this clinical trial was achieved (Table S1). Further, the inter-observer agreement between the two independent sonographers for HyCoSy readings was excellent (Kappa 0.946).
Table 2. Fallopian tube patency test results of HyCoSy and LSC.
| LSC | HyCoSy | |||
|---|---|---|---|---|
| Occluded | Patent | Uncertain† | Total | |
| Occluded | 79 | 18 | 0 | 97 |
| Patent | 8 | 116 | 1 | 125 |
| Uncertain | 0 | 0 | 0 | 0 |
| Total | 87 | 134 | 1 | 222 |
The data represents the number of fallopian tubes, including the left and right fallopian tubes. †, cases classified as “uncertain” were excluded from the diagnostic efficacy analysis. HyCoSy, Hysterosalpingo-Contrast Sonography; LSC, laparoscopy.
False-positive and false-negative results
A total of 26 fallopian tubes showed discordant diagnoses between HyCoSy and LSC. The overall false-positive rate and false-negative rate were 3.62% (8/221) and 8.14% (18/221), respectively. A subgroup analysis was conducted to identify clinical and ultrasound-related factors associated with diagnostic inconsistency. Compared with the true-positive group, the false-positive group had a significantly higher proportion of patients with a history of ectopic pregnancy and with ovaries positioned adjacent to the uterus (P=0.034, P=0.005). However, the incidence of contrast intravasation was significantly higher in the false-negative group than the true-negative group (P=0.046) (Table 3).
Table 3. Clinical and ultrasound factors affecting false-positive and false-negative results.
| Factors | Positive | Negative | |||||
|---|---|---|---|---|---|---|---|
| True | False | P value | True | False | P value | ||
| Age (years) | 30.46±4.34 | 29.88±2.67 | 0.714 | 31.12±4.39 | 31.44±3.33 | 0.410 | |
| BMI (kg/m2) | 22.90±3.47 | 21.14±2.97 | 0.176 | 22.17±3.18 | 22.25±3.99 | 0.087 | |
| Infertile type | 0.485 | 0.259 | |||||
| Primary | 41 (51.90) | 5 (62.50) | 68 (58.62) | 8 (44.44) | |||
| Secondary | 38 (48.10) | 3 (37.50) | 48 (41.38) | 10 (55.56) | |||
| Infertility duration (years) | 3.43±3.20 | 2.00±1.58 | 0.221 | 3.29 ± 2.66 | 3.00±2.40 | 0.967 | |
| History of pelvic surgery | 0.379 | 0.154 | |||||
| No | 52 (65.82) | 4 (50.00) | 78 (67.24) | 9 (50.00) | |||
| Yes | 27 (34.18) | 4 (50.00) | 38 (32.76) | 9 (50.00) | |||
| History of ectopic pregnancy | 0.034 | 0.070 | |||||
| No | 75 (94.94) | 6 (75.00) | 110 (94.82) | 15 (83.33) | |||
| Yes | 4 (5.06) | 2 (25.00) | 6 (5.17) | 3 (16.67) | |||
| Uterine position | 0.756 | 0.952 | |||||
| Anteposition | 63 (79.75) | 6 (75.00) | 81 (69.83) | 12 (66.67) | |||
| Horizontal position | 0 | 0 | 5 (4.31) | 1 (5.56) | |||
| Retroposition | 16 (20.25) | 2 (25.00) | 30 (25.86) | 5 (27.78) | |||
| Side of fallopian tube | 0.528 | 0.612 | |||||
| Left-side | 40 (50.63) | 5 (62.50) | 57 (49.14) | 10 (55.56) | |||
| Right-side | 39 (49.38) | 3 (37.50) | 59 (50.86) | 8 (44.44) | |||
| Ovary position | 0.005 | 0.055 | |||||
| Distant | 64 (81.01) | 3 (62.50) | 103 (88.79) | 13 (72.22) | |||
| Adjacent | 15 (18.99) | 5 (37.50) | 13 (11.21) | 5 (27.78) | |||
| Contrast intravasation | 0.809 | 0.046 | |||||
| No | 56 (70.89) | 6 (75.00) | 90 (77.59) | 10 (55.56) | |||
| Yes | 23 (29.11) | 2 (25.00) | 26 (22.41) | 8 (44.44) | |||
Data are expressed as mean ± standard deviation, or n (%). BMI, body mass index.
Safety results
Based on the study protocol provisions, all 120 enrolled participants were included in the safety analysis. Among the 120 participants, 91 (75.83%) reported 159 TEAEs, while 35 (29.17%) reported 49 AEs related to the investigational drug. Specifically, 32 participants (26.67%) reported 45 mild AEs, and four (3.33%) reported four moderate AEs; no serious AEs were reported. Following LSC, 105 (87.50%) of 112 participants experienced 246 TEAEs, all of which were unrelated to the investigational drug. The overall incidence of AEs following HyCoSy was lower than that following LSC. No participants withdrew from the study due to TEAEs. By the end of the trial, all AEs had resolved or stabilized. No deaths, serious or unexpected AEs occurred during the study.
Among the participants who underwent HyCoSy, the commonly reported TEAEs (incidence ≥5%) included hypogastralgia (44.17%), contrast intravasation (26.67%), and abdominal pain (19.17%). Among the participants who underwent LSC, the commonly reported TEAEs included vaginal bleeding (60.00%), incision site pain (40.83%), and anemia (21.67%), all of which were unrelated to the investigational drug (Table S2).
In terms of pain, approximately one-third of the patients (36/112) considered HyCoSy a painless surgery, and only 1.79% (2/112) experienced severe pelvic pain (as indicated by a score of 8–10 on the VAS). After resting for 15 to 30 minutes, all these patients experienced pain relief. Table 4 shows the pain perception of patients with different levels of patency. Pain levels differed significantly among patients with varying degrees of tubal patency (P=0.012).
Table 4. Pain perceptions of participants with different degrees of patency.
| Tubal patency | Pain perception | ||||
|---|---|---|---|---|---|
| Absent | Mild | Moderate | Severe | Total | |
| Bilateral patency | 20 | 25 | 0 | 0 | 45 |
| Monolateral patency | 10 | 24 | 1 | 0 | 35 |
| Bilateral occlusion | 6 | 20 | 4 | 2 | 32 |
| Total | 36 | 69 | 5 | 2 | 112 |
The pain includes hypogastralgia and abdominal pain.
Discussion
We conducted a multicenter, prospective, blinded clinical diagnostic trial, with a pre- and post-self-controlled design, using LSC as the gold standard, to evaluate the efficacy and safety of perfluoropropane human serum albumin microspheres for HyCoSy in assessing tubal patency. The main study findings were as follows: (I) as expected, HyCoSy with perfluoropropane human albumin microspheres demonstrated accurate diagnostic performance, confirming its effectiveness in the diagnosis of tubal patency; (II) in cases of inconsistent diagnosis, the false-negative results were mainly related to contrast intravasation, while the false-positive results were mainly related to the position of the ovaries and a history of ectopic pregnancy; and (III) in terms of drug safety, the most common AEs were pain and contrast intravasation, which did not require special treatment, and resolved quickly after completion of the procedure.
The use of ultrasound contrast agents in tubal patency assessment continues to evolve. The foam agent ExEm-foamTM used for hysterosalpingo-foam sonography (HyFoSy) is registered for clinical use. Recent studies suggest that HyFoSy may be as accurate as hysterosalpingography in diagnosing tubal patency (16); however, this method has certain practical limitations. For example, foam preparation requires manual mixing, which may lead to inconsistent bubble consistency, and its contrast duration is usually shorter than that of stable ultrasound microbubble contrast agents, which may limit its examination time. Consequently, compared to HyCoSy, HyFoSy has not been widely adopted in clinical practice. With the emergence of second-generation ultrasound contrast agents and advances in ultrasound imaging technology, HyCoSy has become a preliminary diagnostic method for assessing fallopian tube patency (9). However, at present, there is a lack of prospective clinically validated ultrasound contrast agents for HyCoSy. Therefore, we conducted a prospective, double-blind, multicenter, phase III trial in accordance with Good Clinical Practice criteria to provide the highest level of clinical validation for HyCoSy.
The diagnostic performance of HyCoSy confirmed by this rigorous trial is compelling. Our study demonstrated an overall accuracy of perfluoropropane human albumin microspheres use in HyCoSy for evaluating tubal patency was 88.24%, with a sensitivity of 81.44%, and a specificity of 93.55%. A direct comparison of our results with published data for SonoVue (accuracy 85.3%, sensitivity 85.7%, specificity 85.1%) (17) revealed the distinct profile of our agent. Perfluoropropane human albumin microspheres achieved a higher overall accuracy and a substantially superior specificity, while maintaining a comparable sensitivity. This superior specificity is of paramount clinical importance, as it significantly reduces the risk of false-positive diagnoses of tubal occlusion, thereby preventing unnecessary referrals for invasive confirmatory procedures such as LSC. The strong agreement with the laparoscopic gold standard further affirms the reliability of this agent.
The inherent properties of the agent facilitate its excellent diagnostic performance. The use of pre-prepared freeze-dried microsphere technology ensures the stability and controllability of the perfluoropropane human albumin microspheres. Compared with hand-cranked foam agents, this agent is more stable and uniform, which can effectively improve the quality of ultrasonic imaging and reduce the poor imaging quality or incomplete examination caused by the rapid disappearance of the contrast agent. In addition, due to its high molecular weight, low solubility, and dispersion, it can undergo relaxation resonance motion in the fallopian tube cavity, generating linear and nonlinear echoes to improve resolution with surrounding tissues, thereby clearly displaying the morphology of the uterine cavity and fallopian tubes.
However, a certain proportion of false-positive and false-negative results were observed in the HyCoSy assessment of tubal patency. The subgroup analysis identified several key factors influencing the diagnostic concordance between HyCoSy and LSC. Previous ectopic pregnancy surgery may cause structural changes in the fallopian tubes, leading to contrast flow resistance or the formation of abnormal channels that interfere with image interpretation. In addition, when the ovary and the uterine horn are close together, the fallopian tube appears as a tortuous line. An abnormal tubal shape and intestinal gas interference may lead to difficulties in displaying the fallopian tube, thereby affecting the evaluation of tubal patency and resulting in false-positive results (18,19). Conversely, when contrast intravasation occurs, the images may show patchy, striped, or branched shadows that can overlap with the course of the fallopian tubes, increasing the difficulty of analyzing the contrast image, potentially leading to misjudgment (20). He et al. found that early, cornual, and massive intravasation are major risk factors affecting diagnostic image quality, and represent one of the primary causes of false-negative results in ultrasound assessment of tubal patency (21).
Our results indicate that HyCoSy exhibits an “optimistic” bias, with a higher false-negative rate (8.14%) than a false-positive rate (3.62%). While the high specificity reduces unnecessary referrals for LSC, this bias increases the risk of missing true tubal obstruction, potentially delaying appropriate infertility management. The primary factor leading to false-negative results was contrast intravasation, with high-risk factors including a thin endometrium, a history of curettage, or prior fallopian tube surgery (22). Therefore, in clinical practice, a more nuanced approach is warranted for patients with high-risk profiles. We make the following recommendations: (I) for high-risk patients, avoid overfilling the ballon, and inject the contrast agent slowly and uniformly to reduce uterine pressure. (This is the most critical technical point); (II) during image interpretation, pay close attention to any signs of intravasation in high-risk patients, and use multiple imaging modes such as two-dimensional and real-time three-dimensional modes to avoid misinterpreting these artifacts as tubal patency; and (III) if intravasation occurs or findings remain equivocal, consider short-term re-examination or alternative diagnostic methods to minimize the risk of delayed treatment due to false-negative results.
In terms of drug safety, since the National Medical Products Administration approved the use of this drug for left ventricular contrast-enhanced ultrasound in China in 2019 (National Drug Approval Number S20191000), no serious AE reports or deaths have been reported during its clinical use, indicating that it is safe for clinical application. However, during the HyCoSy examination, the contrast agent is only used inside the uterine cavity, passing through the fallopian tubes into the pelvic cavity, where its residence time is very short; thus, the associated risk is relatively low. In this study, the common AEs included pain and contrast agent intravasation, and no deaths, or serious or unexpected AEs occurred. Consistent with previous studies, all the AEs were resolved at the end of the trial (23). Consistent with our findings, research has shown that pain is not caused by the contrast agent itself, but rather by uterine expansion resulting from intrauterine catheterization and balloon dilation, and that fallopian tube patency is strongly correlated with pain (23). Regarding contrast agent intravasation, its incidence in our study was consistent with studies using sulfur hexafluoride microbubble contrast agent, which reported a range of 13.04–38.94% (20,22). In our study, contrast agent intravasation did not require special treatment and resolved quickly (within 0–48 minutes), and did not affect the safety of the participants.
To the best of our knowledge, this was the first multicenter, prospective, double-blind phase III study to evaluate the value of a specific ultrasound contrast agent in HyCoSy, leading to its regulatory approval. This distinguishes it from previous feasibility studies and provides the highest level of evidence for its clinical application. To maintain consistency in the pelvic environment of the participants, the maximum interval between HyCoSy and LSC was set as one menstrual cycle. Additionally, to ensure the reliability of the LSC results, a blinded design was adopted so that the laparoscopic doctors remained blinded to the HyCoSy results before surgery, thereby increasing the scientific rigor and credibility of our data.
This study had several limitations. First, unlike LSC, HyCoSy does not require anesthesia during the examination, as it is easy to operate, non-invasive, and well tolerated. However, some patients may experience false-positive results due to tubal spasms induced by heightened tension and discomfort while in a conscious state. Second, all the enrolled patients had clinical indications for LSC, suggesting a population with relatively complex pelvic conditions. This might have influenced the spectrum of disease and might limit the generalizability of the diagnostic accuracy of this study to a broader infertility population. Third, the primary analysis used the fallopian tube as the unit of analysis, which is standard in tubal patency studies but does not account for within-patient correlation between two tubes. Future studies could use statistical methods such as generalized estimating equations to adjust for the clustering effect in per-tube analyses.
Conclusions
The results of this study indicate that perfluoropropane human albumin microspheres are an effective ultrasound contrast agent for HyCoSy in tubal patency evaluation, demonstrating good diagnostic accuracy compared to the gold standard, LSC. Additionally, this ultrasound contrast agent was well tolerated—no serious AEs were observed—and is thus suitable for clinical application.
Supplementary
The article’s supplementary files as
Acknowledgments
We would like to thank all the study participants and the study staff. We would also like to thank all the participating investigational sites and the principal investigators at these sites: The First Affiliated Hospital of Sun Yat-sen University, The Third Affiliated Hospital of Guangzhou Medical University, Xiamen Women and Children’s Hospital Affiliated to Xiamen University, Hunan Provincial Women and Children’s Hospital, Nanfang Hospital of Southern Medical University, Jiangnan University Obstetrics and Gynecology Hospital, Zhongnan Hospital of Wuhan University, Jieyang People’s Hospital, Nanchang Reproductive Hospital, Women’s Hospital of Nanjing Medical University, and Tongji University Tenth People’s Hospital. We would also like to thank Xiamen Lizhuo Pharmaceutical Co., Ltd. in China, for its constructive and valuable support.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by Independent Ethics Committee or Independent Review Board of all 11 participating clinical research centers according to national or local regulations and informed consent was taken from all individual participants.
Footnotes
Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1506/rc
Funding: This work was supported by the Guangzhou Municipal Science and Technology Project (grant No. 202201011238), the National Natural Science Foundation of China (grant Nos. 81571687, 82171938 and 82202156).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1506/coif). X.Z. is a full-time employee of Xiamen Lizhuo Pharmaceutical Co., Ltd. The other authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1506/dss
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Data Availability Statement
Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1506/dss